Centrifugal fermentation process

ABSTRACT

The present invention comprises a novel culture methods and devices in which living cells or subcellular biocatalysts are immobilized by the opposition of forces. The immobilized cells or biocatalysts may be attached to support complexes that add to the resultant vector forces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationsNos. 60/070,304; 60/070,251; 60/070,270; 60/070,133; 60/070,283;60/070,078; 60/070,301 all filed Dec. 31, 1997, and this application isa continuation-in-part of U.S. patent application Ser. No. 09/115,109,filed Jul. 13, 1998, now U.S. Pat. No. 6,133,019, which is acontinuation-in-part of U.S. patent application Ser. No. 08/784,718,filed Jan. 16, 1997, now U.S. Pat. No. 5,821,116, which is a division ofU.S. patent application Ser. No. 08/412,289, filed Mar. 28, 1995, nowU.S. Pat. No. 5,622,819.

FIELD OF THE INVENTION

The present invention relates to an improved method and apparatus forthe continuous culture of biocatalysts. More particularly, the presentinvention relates to a method and apparatus for culturingmicro-organisms, or plant or animal cells, or subcellular cellcomponents as three-dimensional arrays immobilized in centrifugal forcefields which are opposed by liquid flows. The present invention allowsthe maintenance of extremely high density cultures of biocatalysts andmaximizes their productivity.

BACKGROUND OF THE INVENTION

The term “fermentation” as used herein means any of a group of chemicalreactions induced by living or nonliving biocatalysts. The term“culture” as used herein means the suspension or attachment of any suchbiocatalyst in or covered by a liquid medium for the purpose ofmaintaining chemical reactions. The term “biocatalysts” as used herein,includes enzymes, vitamins, enzyme aggregates, immobilized enzymes,subcellular components, prokaryotic cells, and eukaryotic cells. Theterm “centrifugal force” means a centripetal force resulting fromangular rotation of an object when viewed from a congruently rotatingframe of reference.

The culture of microbial cells (fermentation) or animal and plant cells(tissue culture) are central to a multiplicity of commercially-importantchemical and biochemical production processes. Living cells are employedin these processes as a result of the fact that living cells, usinggenerally easily obtainable starting materials, can economicallysynthesize commercially-valuable chemicals.

Fermentation involves the growth or maintenance of living cells in anutrient liquid media. In a typical batch fermentation process, thedesired micro-organism or eukaryotic cell is placed in a defined mediumcomposed of water, nutrient chemicals and dissolved gases, and allowedto grow (or multiply) to a desired culture density. The liquid mediummust contain all the chemicals which the cells require for their lifeprocesses and also should provide the optimal environmental conditionsfor their continued growth and/or replication. Currently, arepresentative microbial cell culture process might utilize either acontinuous stirred-tank reactor or a gas-fluidized bed reactor in whichthe microbe population is suspended in circulating nutrient media.Similarly, in vitro mammalian cell culture might employ a suspendedculture of cells in roller flasks or, for cells requiring surface,attachment, cultures grown to confluence in tissue culture flaskscontaining nutrient medium above the attached cells. The living cells,so maintained, then metabolically produce the desired product(s) fromprecursor chemicals introduced into the nutrient mixture. The desiredproduct(s) are either purified from the liquid medium or are extractedfrom the cells themselves.

Examples of methods employing fermentations of cells growing in eitheragitated aqueous suspension or with surface attachment are described,for example, in U.S. Pat. Nos. 3,450,598; 3,843,454; 4,059,485;4,166,768; 4,178,209; 4,184,916; 4,413,058; and 4,463,019. Furtherreference to these and other such conventional cell culturing techniquesmay be found in such standard texts as Kruse and Patterson, TissueCulture Methods and Applications, Academic Press, New York, 1977; andCollins and Lyne's Microbiological Methods, Butterworths, Boston, 1989.

There are a number of disadvantages inherent in such typicalfermentation processes. On a commercial scale, such processes requireexpensive energy expenditures to maintain the large volumes of aqueoussolution at the proper temperature for optimal cell viability. Inaddition, because the metabolic activity of the growing cell populationcauses decreases in the optimal levels of nutrients in the culture mediaand causes changes in the media pH, the process must be continuouslymonitored and additions must be made to maintain nutrient concentrationand pH at optimal levels.

In addition, the optimal conditions under which the desired cell typemay be cultured are usually near the optimal conditions for the growthof many other undesirable cells or microorganisms. Extreme care andexpense must be taken to initially sterilize and to subsequently excludeundesired cell types from gaining access to the culture medium. Next,such fermentation methods, particularly those employing aerobicorganisms, are quite often limited to low yields of product or low ratesof product formation as a result of the inability to deliver adequatequantities of dissolved oxygen to the metabolizing organism. Finally,such batch or semi-batch processes can only be operated for a finitetime period before the buildup of excreted wastes in the fermentationmedia require process shutdown followed by system cleanup,resterilization, and a re-start.

The high costs associated with the preparation, sterilization, andtemperature control of the large volumes of aqueous nutrient medianeeded for such cultures has led to the development of a number ofprocesses whereby the desired cell type or enzyme can be immobilized ina much smaller volume through which smaller quantities of nutrient mediacan be passed. Cell immobilization also allows for a much greatereffective density of cell growth and results in a much reduced loss ofproductive cells to output product streams. Thus, methods and processesfor the immobilization of living cells are of considerable interest inthe development of commercially valuable biotechnologies.

An early method for the immobilization of cells or enzymes involved theentrapment of such biocatalysts on or within dextran, polyacrylamide,nylon, polystyrene, calcium alginate, or agar gel structures. Similarly,the ability of many animal cells to tenaciously adhere to the externalsurface of spherical polymeric “microcarrier beads” has likewise beenexploited for the immobilization of such cells. These gel- orbead-immobilization methods effectively increase the density of thebiocatalyst-containing fraction, thereby effectively trapping thesestructures in the lower levels of relatively slow-flowing bioreactorchambers. Such gel-entrapment or microcarrier-immobilized methods aretaught, for example, in U.S. Pat. Nos. 3,717,551; 4,036,693; 4,148,689;4,189,534; 4,203,801; 4,237,033; 4,237,218; 4,266,032; 4,289,854;4,293,654; 4,335,215; and 4,898,718. More background information on cellimmobilization techniques is discussed in Chibata, et al., “ImmobilizedCells in the Preparation of Fine Chemicals”, Advances inBiotechnological Processes, Vol. I, A.R. Liss, Inc., New York, 1983. Seealso Clark and Hirtenstein, Ann. N.Y. Acad. Sci. 369, 33-45 (1981), formore background information on microcarrier culture techniques.

These immobilization methods suffer from a number of drawbacks. First,such entrapment of cells within gels has been shown to interfere withthe diffusion of gases (particularly oxygen and carbon dioxide) into andout of the cell environment, resulting in either low cell growth(reduced oxygen input) or gel breakage (high internal CO₂ pressure). Inaddition, the poor mechanical properties and high compressibility ofgel-entrapment media lead to unacceptably high pressure problems inpacked bed bioreactors. Similarly, the crushing of microcarrier beadsand the destruction of attached cells by hydraulic shear forces inagitated tank bioreactors (necessary to increase gas exchange) leads toreduced viability and productivity.

Another method for the immobilization of living cells or enzymescurrently in use involves the use of packed-bed bioreactors. In thesemethods, free cells or cells bound to microcarrier beads are suspendedin a rigid or semi-rigid matrix which is placed within a culturebioreactor. The matrix possesses interstitial passages for the transportof liquid nutrient media into the bioreactor, similarly disposedpassages for the outflow of liquid media and product chemicals, andsimilar interstitial passages through which input and output gases mayflow. Bioreactors of this type include the vat type, the packed-columntype, and the porous ceramic-matrix type bioreactor. Such methods aretaught, for example, in U.S. Pat. Nos. 4,203,801; 4,220,725; 4,279,753;4,391,912; 4,442,206; 4,537,860; 4,603,109; 4,693,983; 4,833,083;4,898,718; and 4,931,401.

These methods of immobilization all suffer from a number of problems,particularly when scaled up to production size. First of all, suchbioreactors are subject to concentration gradients. That is, thebiocatalysts nearer the input nutrient liquid feed see higher substratelevels than those farther downstream. Conversely, those biocatalystsfarther from the input liquid stream (and closer to the exit liquidport) see increased concentrations of waste products and often suffersuboptimal environmental conditions, such as a changed pH and/or lowereddissolved oxygen tension. Next, such bioreactors are particularlysusceptible to the “bleeding” of biocatalysts detached from the matrix(or released by cell division), with the result that output ports becomeclogged with cells and/or debris. The result is an unacceptable pressuredrop across the bioreactor which causes further deterioration ofproduction. Finally, such vertical packed-bed bioreactors in which glassor other microcarrier beads are packed subject the lower portion of thebed to the weight of those beads above, with the inevitable result thatboth beads and cells are crushed by the sheer weight and number of beadsneeded for production-scale columns.

A more recently-developed class of methods for cell immobilizationinvolves the confinement of the desired cells between two syntheticmembranes. Typically, one membrane is microporous and hydrophilic and incontact with the aqueous nutrient media, while the opposing membrane isultraporous and hydrophobic and in contact with a flow of air or anoxygen-enriched gas. Such processes thus provide the cells with anenvironment in which nutrient liquid input and waste liquid output canoccur through channels separate from the cell-containing space andsimilarly provide gaseous input and output through similarly disposedchannels, again separate from the cell-containing space. Embodiments ofmethods of this class have utilized stacks of many flat membranesforming a multiplicity of cell compartments, have utilized series ofsynthetic membrane bags, one within the other, and have utilizedspirally-wound membrane configurations. Such methods are taught, forexample, in U.S. Pat. Nos. 3,580,840; 3,843,454; 3,941,662; 3,948,732;4,225,671; 4,661,455; 4,748,124; 4,764,471; 4,839,292; 4,895,806; and4,937,196.

Unfortunately, there are a number of problems with such methods,particularly for any commercial, large-scale usage. First, such devicesin which a multiplicity of membranes are stacked in series are quitecostly to manufacture and are extremely difficult to correctly assemble.Next, the requirement that the membrane which separates the nutrientchannels from the immobilized cells be hydrophilic necessarily resultsin cell attachment across pores, and/or pore clogging by insolubles ineither the nutrient feed or waste output liquids which wet thismembrane. The result is the development over time of “dead pockets”where cell growth cannot occur. This situation greatly reduces theeffective cell concentration and lowers product yield. Finally, thesemethods involve devices with a large number of inlet and outlet portsand external fittings which substantially increase both cost and theprobability that leakage and contamination will occur.

Another class of methods for cell immobilization involves the employmentof capillary hollow fibers (usually configured in elongated bundles ofmany fibers) having micropores in the fiber walls. Typically, cells arecultured in a closed chamber into which the fiber bundles are placed.Nutrient aqueous solutions flow freely through the capillary lumena andthe hydrostatic pressure of this flow results in an outward radialperfusion of the nutrient liquid into the extracapillary space in agradient beginning at the entry port. Similarly, this pressuredifferential drives an outward flow of “spent” media from the cellchamber back into the capillary lumena by which wastes are removed.Cells grow in the extracapillary space either in free solution or byattachment to the extracapillary walls of the fibers. Typically, oxygenis dissolved into the liquid fraction of the extracapillary space bymeans of an external reservoir connected to this space via a pumpmechanism. Waste products in the intracapillary space may be removed byreverse osmosis in fluid circulated outside of the cell chamber. Suchmethods are taught, for example, by U.S. Pat. Nos. 3,821,087; 3,883,393;3,997,396; 4,087,327; 4,184,922; 4,201,845; 4,220,725; 4,442,206;4,722,902; 4,804,628; and 4,894,342. There are a number of difficultieswith the use of methods based on capillary hollow fiber cellimmobilization methods.

Cracauer et al. (U.S. Pat. No. 4,804,628) have extensively documentedthese difficulties. These difficulties include: (1) an excessivepressure drop through the fiber assembly (The fragile nature of thefibers results in complete breakdown if fiber of production-scale lengthis required.); (2) the occurrence of adverse chemical gradients withinthe cell chamber (Gradients of nutrients and waste products often occurin such chambers.); (3) the formation of anoxic pockets and discretedisadvantageous microenvironments within the cell chamber (Because ofthe inaccessibility of liquids, gases, and cells to all portions of thefiber bundle as a result of their design, not all areas of the cellchamber are equally effective in cell production.); and (4) eithermass-transfer limitations in nutrient feed or limitations in productoutput increase with time. (As cells grow to higher densities, they tendto self-limit the capacities of the hollow fiber chambers (see Col. 1,lines 53-66, of U.S. Pat. No. 4,804,628)).

Another class of methods for the mass culture of living cells involvesthe use of fluidized bed bioreactors. The excellent mixingcharacteristics and fluid dynamics of this type of mass culture havefound usage in both microbial and bead-immobilized animal cell culture.The major disadvantage of fluidized bed methods, and particularly avariant called airlift fermentors, results from the necessity ofbubbling air or oxygen through the bioreactor and the resultant presenceof a gas-liquid interface throughout the bioreactor volume. Firstly, thepresence of gas bubbles in the flowing liquid disrupts the fluiddynamics which provide the initial advantages of fluidized beds (uniformparticle suspension). Next, protein foaming, cell destruction, and thedenaturation of nutrients and products occurs at the large gas-liquidinterface. Finally, cell washout is almost inevitable in continuousoperation, particularly with animal cell culture.

Another class of methods for mass cell culture is known as dual axis,continuous flow bioreactor processing. Such methods are taught by, forexample, U.S. Pat. Nos. 5,151,368, 4,296,882, and 4,874,358. In thisclass of bioreactor, rotation of the bioreactor chamber about an axisperpendicular to the vertical axis is utilized in order to effectinternal mixing of the bioreactor contents while rotation about thevertical axis confines grossly particulate matter at radial distancesfar from the vertical axis of rotation. Input nutrient liquids and gasesare supplied by concentric flexible conduits into the bioreactor andoutput liquids and gases are removed by similar flexible conduitsconcentric with the input tubings. While the intended purpose ofbioreactors of this class is to allow continuous flow of liquid into andout of a bioreactor chamber in which a combination of solids and liquidsis suspended and mixed, such processes are limited to rotational speedsat which effective mixing can occur without appreciable negation bycentrifugal forces. As a result, methods of this class are ineffectivein the immobilization of low mass micro-organisms, particularly thoserequiring gaseous nutrients and producing waste gas products. Othersimilar centrifugal liquid processing apparati are disclosed in U.S.Pat. Nos. 4,113,173, 4,114,802, 4,372,484, and 4,425,112. In each ofthese latter references, liquid flow through a centrifugal chamber issupplied by flexible tubing extending through the rotational axis.

Another type of bioreactor called a “Nonhomogeneous Centrifugal FilmBioreactor” intended for aerobic cell culture is taught by U.S. Pat. No.5,248,613. The object of the method is to maximize the “entrainment ofthe maximum amount of the gaseous phase into the liquid phase” bycausing the formation of a thin liquid film to contact the gas phase andfurther, to centrifugally generate small liquid droplets which fallthrough a relatively stationary gas phase back into the recirculatedbulk liquid phase. There are a number of problems associated with abioreactor design of this type. First of all, it is a “batch” process.That is, the nutrient liquid phase gradually is depleted of itscomponents while liquid metabolic wastes build up, necessitating alimited culture time. Secondly, the scale of such a bioreactor islimited by the quantity of nutrient gas (such as oxygen) which can bedissolved in the various gas-liquid transfer regions. In the limit, themaximum gas transfer obtainable at atmospheric pressure will determinethe maximum cell “load” which can be carried by the bioreactor system.Next, the lack of any provision for the removal of waste gases (such ascarbon dioxide) will result in disruption of both bulk liquid pH as wellas cellular productivity as culture periods extend to longer times.Finally, it is extremely doubtful that accelerated productive cell losscould be avoided if animal cells were subjected to passage through ahigh flow-rate, thin-film liquid region where cell-disruptingsurface-tension forces are maximal, and where there is limited nutrientavailability due to the presence of maximum aerobicity.

A final method for the mass immobilization of living cells called“Continuous Centrifugal Bioprocessing” has been taught by Van Wie, etal. (U.S. Pat. No. 4,939,087). In this method cells are “captured” by avelocity gradient in a centrifugal field in order to maintain a culturein a revolving bioreactor chamber into which and out of which liquidflows are pumped. The basic idea upon which the invention of Van Wie etal. is based was first postulated by Lindahl in 1948 (Lindahl, P. E.(1948) Nature (London) 161, 648-649) and a U.S. Patent awarded in thesame year to MacLeod (U.S. Pat. No. 2,616,619). More recently, BeckmanInstruments has developed analytical devices called “CentrifugalElutriation Systems” based on the general principles of what is termed“Counterflow Centrifugation.” The particle and fluid dynamic theory uponwhich these devices were constructed and refined has been mostcompletely discussed by Sanderson and Bird (Sanderson, R. J. and Bird,K. E. (1977) Methods in Cell Biology, 15, 1-14). As is shown in FIG. 1,the basic idea is to suspend a particle in a spinning bioreactorchamber, which as a consequence of its rotation, imparts a “centrifugal”force to the particle which would normally cause the particle to migrateto longer centrifugal radii. Liquid flow is introduced into theperiphery of the spinning chamber (and withdrawn at shorter radii) inorder to impart an opposing force which counteracts that of thecentrifugal field. The result is that the particle is immobilized at aparticular radial distance in a liquid flow. The essence of Sandersonand Bird's mathematical analysis of the particle and fluid dynamics ofthis process are displayed in FIG. 2. As do all theoretical discussionsof centrifugation theory, Sanderson and Bird's analysis begins with theapplication of Stoke's Law at low Reynolds numbers, an expression whichgoverns the motion of a particle moving through an incompressible fluid(Eqn. 1). Briefly, the law states that the sedimentation velocity (SV)of a non-deformable particle moving through a stationary liquid underthe influence of a centrifugal field is proportional to the square ofthe angular velocity (_(—) ²r) of the rotating system at radius rmultiplied by the following expression: the square of the effectivediameter of the particle (d) multiplied by the difference between thedensity of the particle and the density of the liquid (_p−_m) divided bythe product of the liquid viscosity (_) and the “shape constant” of theparticle (k, its deviation from sphericity). As was recognized first byLindahl, the same equation applies to a stationary particle in a movingliquid flow. The analysis of Sanderson and Bird led to the derivation ofEqn. 2, an expression which states that “there is a radius r_(x)(defined by evaluation of Eqn. 2) at which a particle is immobilized ina liquid flowing at velocity (V) in an centrifugal field (the parametersof Eqn. 2 are those defined above where (_p−_m) has been replaced by(_′). These authors further conclude that the contribution of thecoriolis force to the net motion of the particle is negligible since itis limited to a tangential plane.

This theory, when applied to Centrifugal Elutriation, (as it was bySanderson and Bird and by developers at Beckman Instruments) can beutilized in the short term for the separation of cells of different sizeand/or density. Unfortunately, this theory is completely inapplicable tolong-term immobilization of cells or biocatalysts (as is implicit inU.S. Pat. No. 4,939,087) since the theoretical basis is incorrect. As isshown in FIG. 3, there is an additional force acting on the suspendedparticle which must be taken into account, particularly when theparticle is to be immobilized over long time periods (as would be thecase in fermentations). This additional force is a result of theparticle's mass. Whereas micro-organisms or animal cells are quite lightin weight, their mass is non-zero. Consequently, gravity will have asignificant effect on the particle, and this effect will increase withtime. This is shown graphically in FIG. 4, where it is shown that thereis not a simple description of a radial distance where a particle in anapplied centrifugal field can be immobilized in a flowing liquid sincethe derivation has neglected to consider the effect of gravity on themass of the particle. The result of this “deviation from theory” isevident in centrifugal elutriation experiments which require prolongedseparation times and is shown graphically in FIG. 5. Over longer timeperiods, the weight of the suspended particles (shown in FIG. 5 as darkcircles in a circular cross-section of a biocatalyst immobilizationchamber) will cause these particles to settle to the lowest regions ofthe biocatalyst immobilization chamber, disrupting the balance of forceswhich initially suspended them in the chamber. Further, the“aggregation” of these particles into a larger “particle” with virtuallythe same density as the individual particles results in an increasedcentrifugal effect which causes the aggregates to migrate to longerradii, eventually clogging the liquid input port.

There are several additional disadvantages to the “ContinuousCentrifugal Bioprocessing” art taught by U.S. Pat. No. 4,939,087. Firstof all, the method is seriously limited by its design (which includesclockwork-like gear assemblies and moving flexible tube inputs andoutput lines) to low-speed operation. This means that the method couldbe used neither for the culture of low-mass micro-organisms nor largescale cultures of high mass cells in which the required liquid flowrates for adequate nutrition of the cultures would require rotationalrates greatly in excess of those allowable by the apparatus in order toprovide a counter-acting “centrifugal” force. Next, the method by whichgaseous air/carbon dioxide is introduced into the bioreactor chamber (agas-permeable flexible tube in contact with similar flexible tubes whichtransport input and output liquid flows) will greatly limit the scale ofthe apparatus since, very rapidly, the required aeration to support cellviability will be limited by the physical pressure and diffusion limitsof the flexible tubing. Finally, the apparatus of Van Wie, et al. makesno provision for the vigorous outgassing of, for example, carbon dioxidewhich will occur as a result of cell metabolism. The metabolicallyproduced gases will: (1) greatly disrupt the input gas exchangenecessary for viability by limiting the liquid surface area in contactwith the gas-permeable tubing; (2) greatly limit the efficient functionof the pumping mechanisms necessary for liquid flow into and out of theapparatus; (3) result in the growth of gas pockets in the upper portionsof the horizontally rotating bioreactor chamber with a resultantdecrease of effective bioreactor volume and cell loss by bubbleentrainment; and (4) result in serious rotor balance problems.

The prior art demonstrates that while cell immobilization is a greatlydesired method for increasing the productivity of living cells inculture, there are a number of drawbacks associated with each class ofmethod. A central problem of all such culture methods is, as Wrasidlo etal. (U.S. Pat. No. 4,937,196) assert, that “adequate oxygenation of thecultured cells and removal of carbon dioxide has been a limiting factorin the development of more efficient and economical designs” (see Col.1, lines 63-65, of U.S. Pat. No. 4,937,196).

Living cells or bio-catalytic subcellular components are unable toderive any benefit from gaseous oxygen. Living cells or biocatalystsderive benefit solely from oxygen dissolved within the aqueous mediawhich surrounds the particles. In batch fermentations which are commonfor microbial production, the sparging of air or oxygen-enriched gasesthrough the aqueous nutrient media is intended to replace the dissolvedoxygen consumed by the metabolizing cells. In this method, most of thegas exits unused while dissolved oxygen levels are maintained at somevalue. Similarly, the sparging of air (or oxygen) into the nutrientmedia prior to its use in animal cell culture is intended to maintain alevel of dissolved oxygen in the media. While the normal concentrationof oxygen in water varies from about 0.2 to 0.3 mM (depending on suchfactors as pH and ionic strength), it is possible to increase thisconcentration to as much as 0.5 mM by applying approximately twoatmospheres of oxygen pressure over a water solution.

To maintain adequate oxygen concentrations in fermentation media, mostof the prior art has focused on increasing the contact between gas andliquid by: (1) producing a very small bubble size (a function of thesparging frit pore size); (2) using high-speed agitation to increase therate of oxygen entrance into the liquid phase; or (3) using a gaseousoverpressure of one or two atmospheres above the culture medium toincrease dissolved oxygen levels. In the case of animal cell culture,the typical design of animal cell culture chambers has heretofore madeit difficult to consider using overpressures greater than a fraction ofan atmosphere. Thus, the most common method for increasing oxygen levelsemploys gas-permeable membranes or fibers in contact with flowingnutrient liquid to maintain dissolved oxygen levels. Such methods aretaught, for example, by U.S. Pat. Nos. 3,968,035; 4,001,090; 4,169,010;4,774,187; 4,837,390; 4,833,089; and 4,897,359.

There are a number of problems associated with these methods ofincreasing the concentration of dissolved oxygen in nutrient media.First and foremost, nearly all of these methods are unable to increasedissolved oxygen concentrations above that obtainable at atmosphericpressure due to the generally fragile nature of other components of thecell culture process. Next, methods which involve vigorous agitation ofthe liquid-gas mixture to effect increased rates of oxygen dissolutionare not applicable to animal cells, which are quite fragile and caneasily be damaged by hydraulic shear forces. Finally, those methodswhich do apply an increased gaseous overpressure above the culture mediato increase dissolved oxygen concentrations cannot be scaled up muchhigher than approximately 1-2 atmospheres of overpressure before itbecomes impossible to access the cell-containing liquid media for cellharvest or product isolation without destroying the cultured cells.Nevertheless, the teachings of each of the above methods warrantindividual discussion.

U.S. Pat. No. 4,897,359 (issued to Oakley, et al.) discloses a methodfor oxygenating animal cell culture media for subsequent introductioninto cell culture vessels in which an oxygenated gas, at anindeterminate pressure, is passed through a multiplicity ofgas-permeable tubes surrounded by the liquid medium to be oxygenated.While the pressure of the input gas may be above atmospheric pressure,the pressure of the oxygenated exit liquid can be no more thanatmospheric pressure. If the oxygenated exit liquid were aboveatmospheric pressure, it would result in outgassing of the liquid mediumwhen the medium was introduced into the typical cell culture vessel.Such outgassing would also result in bubble formation within the media,which would be extremely deleterious to animal cell viability. Thus, themethod of the invention of Oakley, et al. is useful only in assuringthat the cell culture media possesses the maximum dissolved oxygenconcentration obtainable at atmospheric pressure.

U.S. Pat. No. 4,837,390 (issued to Reneau) discloses a method ofpreservation of living organs (for subsequent transplant) in whichhyperbaric conditions (2 to 15 bars or 29 to 218 pounds per square inch(psi)) are maintained. In the Reneau method, a living organ is placed ina chamber capable of withstanding pressure, and a perfusion liquidcontaining nutrients is pumped into and out of the chamber while agaseous oxygen overpressure is also applied to the chamber. The methoddoes not discuss cell culture or fermentation.

U.S. Pat. No. 4,833,089 (issued to Kojima, et al.) discloses a cellculture method in which a gaseous overpressure of oxygen or air isapplied over a stirred liquid media in which cells are cultured. In thismethod, the pressure limitations of the apparatus (which includesperistaltic pumps, flexible low-pressure pump tubing, and low-pressurefilter apparati) necessarily limit the method to overpressures of0.3-0.7 kg/cm² (approximately 4.3-10 psi). Thus, the concentration ofdissolved oxygen in the media used to bathe the cells is limited tovalues only slightly greater than that obtainable at atmosphericpressure (Col. 4, lines 15-17).

U.S. Pat. No. 4,774,187 (issued to Lehmann) discloses a method for theculture of microbial cells in which a gaseous overpressure is appliedover stirred liquid media in which cells are cultured. In this method,the gaseous overpressure makes it impossible to access the interior ofthe culture compartment without depressurization and cell destruction.Lehman overcomes this problem by raising an overflow line from themedia-containing bioreactor to a height such that the liquid pressure ofthis overflow line equals the gas overpressure. By establishing a siphon(originating in the elevated overflow vessel) connected to the overflowline, one may withdraw liquid or cells from the culture chamber withoutdepressurizing the chamber. Because the typical culture medium isessentially an aqueous solution, the system pressure is limited to theheight of a column of water which would balance the system pressure.Thus, for example, at a system pressure of 37 psi (gauge), a column ofwater approximately 50 feet in height would be required. Thus, from apractical standpoint, the Lehmann method is limited to dissolved oxygenlevels obtainable at 1-2 atmospheres of overpressure.

U.S. Pat. No. 4,169,010 (issued to Marwil) discloses a method forimproved oxygen utilization during the fermentation of single cellprotein in which a gaseous overpressure above a stirred nutrient liquidin a bioreactor containing the growing cells is utilized to increaseoxygen delivery to the growing cells. In this method, the recirculationof cell-free media (lean ferment) obtained by centrifugation of thebioreactor contents is passed back into the bioreactor through anabsorber section containing a gas contacting zone. The gaseousoverpressure is maintained by a gas pressure regulator device whichblocks pressure release or vents the gas in response to a desireddissolved oxygen sensor setting. The patent discloses overpressures ofabout 0.1 to 100 atmospheres (approximately 16.2 to 1485 psi) (Col. 7,lines 28-30, of U.S. Pat. No. 4,169,010). Marwil states that a maximumdesirable gaseous overpressure of 1 to 2 atmospheres is preferable.

Presumably, the reason that a maximum desirable gaseous overpressure of1 to 2 atmospheres is preferable in the Marwil method, and would bedifficult to exceed, arises from the fact that the metabolizing cellsalso release carbon dioxide, a metabolite which must be removed from thenutrient media by gas evolution if cell viability is to be maintained.Gas overpressures greater than 1 to 2 atmospheres utilized to increasedissolved oxygen content would necessarily result in very largedissolved carbon dioxide levels retained within the nutrient media whichcould not be removed until the gaseous overpressure was released. Itshould be noted that carbon dioxide solubility in aqueous solution isapproximately an order of magnitude greater than that of oxygen. Theinability to remove dissolved carbon dioxide from the media while stilldelivering increased oxygen to the media would cause an undesireddecrease in aqueous pH. This decrease in pH is a serious problem of themethod of Marwil. In addition, the method of Marwil is designed solelyfor the continuous harvest of cells; the method cannot be applied to thecontinuous harvest of the aqueous solution which might contain anexcreted cellular product chemical.

U.S. Pat. No. 4,001,090 (issued to Kalina) discloses a method formicrobial cell culture which incorporates a process for improved oxygenutilization which is very similar to that outlined above for Marwil(U.S. Pat. No. 4,169,010). The method of Kalina directly addresses theproblem of carbon dioxide removal mentioned earlier in connection withthe method of Marwil. This problem is eliminated by the inclusion of agas-liquid separator in the fermentor circuit. In the method of Kalina,an oxygenated gas at an unspecified pressure greater than atmospheric isreleased into the fermentation chamber at its bottom (common sparging).However, by means of a backpressure device, the media is maintained atan overpressure of as much as 3 to 3.5 atmospheres (44.1 to 51.5 psi) toprovide both a motive force for the media recirculation, as well as toaid in the removal of excess gas distal to the fermentation zone (Col.4, lines 35-37). The Kalina process relies heavily on the presence ofgas bubbles for the agitation of the media and is suitable solely foruse in microbial cell fermentation. The method could not be applied toanimal cell culture because animal cells are extremely sensitive tohydraulic shear forces and are damaged or destroyed by contact withair-water interfaces such as those encountered in gas bubble-containingmedia.

U.S. Pat. No. 3,968,035 (issued to Howe) discloses a method for the“super-oxygenation” of microbial fermentation media in which the commonsparging of an oxygen-containing gas into the fermentation media isreplaced by the introduction of this gas into an “oxidator” vessel inwhich high-shear agitation is used to reduce the average size of the gasbubbles, thus increasing the available surface area for gas-liquidcontact with the result that maximal dissolved oxygen concentration ismaintained. The fermentation media which has thus been treated is pumpedinto the fermentation reactor while exhausted media from this samesource provides the input to the “oxidator” vessel. The method in Howethus provides a combined liquid and oxygen-enriched gaseous mixture tothe culture chamber; a situation which is inapplicable to animal cellculture for the previously-mentioned reasons.

Because the immobilization of cells or microorganisms requires that acell culture chamber be part of the process system, the recentliterature on cell culture chambers has been examined for comparison.There are a number of cell culture chambers in existence. Many of thesechambers provide for the input and output of a liquid stream, severalhave viewing ports, and all provide a surface upon which cells mayattach or a chamber in which suspended cells may be cultured. Suchmethods are taught, for example, in U.S. Pat. Nos. 3,871,961; 3,753,731;3,865,695; 3,928,142; 4,195,131; 4,308,351; 4,546,085; 4,667,504;4,734,372; 4,851,354; and 4,908,319. In all cases, the operatingpressure of these confinement chambers is one atmosphere (or less).Thus, these chambers are unsuitable for processes in which increaseddissolved oxygen levels are desired, and are necessarily limited tothose dissolved oxygen levels obtainable at atmospheric pressure.

The current state of the art reveals that there are three inter-relatedproblems which plague the economical use of mass cultures of microbes,animal cells, or their subcellular components. First, as is evident fromthe sheer volume of the prior art on cell immobilization, the primaryproblem relates to increasing the density of the cell culture. It isobvious that the economical production of a biological product will bedirectly related to the ability to efficiently culture large aggregatesof the desired cell type. Unfortunately, the drive to increase cellculture density has lead to the evolution of the two secondary problems,the inability to adequately nutrition a high density cell aggregate, andthe inability to supply adequate oxygen to high density aerobic cellpopulations. As cell density is increased, the only method for supplyingadequate liquid nutrient to the aggregate involves increased liquid flowrates which, in all cases in the prior art, eventually limits theoverall scale of the immobilization method. Similarly, as the celldensity increases, the inability to deliver adequate dissolved oxygen(or any other gas) to the cell aggregate is even more of a limitingfactor and severely reduces the scale of the culture.

Accordingly, there remains a need for an apparatus and method forcontinuously culturing, feeding, and extracting biochemical productsfrom either microbial or eukaryotic cells or their subcellularcomponents while maintaining viable, high density aggregates of thesebiocatalysts. In addition, there is a need for a method for the absoluteimmobilization of sample biocatalyst populations which will allow thestudy of various nutritive, growth, and productive parameters to providea more accurate understanding of the inter-relationships between theseparameters and their effects on cell viability and productivity.

SUMMARY OF THE INVENTION

The present invention comprises a novel culture method and apparatus inwhich living cells or subcellular biocatalysts are immobilized withinbioreactor chambers mounted in a centrifugal field while nutrientliquids, without any gas phase(s) in contact with the liquids, areflowed into and out of the bioreactor chambers. The cells orbiocatalysts are ordered into a three-dimensional array of particles,the density of which is determined by the particle size, shape,intrinsic density, and by the selection of combinations of easilycontrollable parameters such as liquid flow rate and angular velocity ofrotation.

According to the present invention, the cells or biocatalysts can beconfined within the bioreactor chambers at a defined volume. Onlyliquids (which may contain dissolved gases) are passed into and out ofthe bioreactor chambers. To cause nutrient liquids to flow through thethree-dimensional array of cells or catalysts in the bioreactorchambers, positive displacement pumps are employed to move the nutrientliquid, at positive hydraulic pressure, through the bioreactor chambers.The confined cells or biocatalysts are unaffected by the resultantincrease in hydraulic pressure as long as high-frequency pressurefluctuations are not present. Thus, fresh, optimal liquid nutrient mediais presented to the confined cells or biocatalysts at all times duringthe process flow while desired cellular products are immediatelyaccessible at the output of the bioreactor chambers.

The present invention can be used to produce high yields of industrialchemicals or pharmaceutical products from biocatalysts such as bacteria,yeasts, fungi, and eukaryotic cells or subcellular organelles, such asmitochondria, or immobilized enzyme complexes. These cells or cellularsubstructures can be either naturally occurring or can be geneticallymanipulated to produce the desired product. The present invention can beoperated in either of two modes: (1) a mode in which nutrient limitationis used to ensure a defined bioreactor bed volume. This mode isapplicable to cultures where desired products are released from theimmobilized biocatalysts and exit the bioreactor in the liquid flow; (2)a mode in which excess nutrient input is used to cause overgrowth of thevolume limitation of the bioreactor. This mode is useful for thecontinual production and outflow of mature cells containing anintracellular product.

Accordingly, it is an object of the present invention to provide amethod and apparatus by which biocatalysts are immobilized withinbioreactor chambers while nutrient liquids are fed into the bioreactorchambers and effluent liquids containing desired metabolic product(s)exit the bioreactor chambers.

It is a further object of the present invention to provide a method andapparatus by which biocatalysts, including living cell populations, maybe immobilized and either aerobic or anaerobic fermentations performedin which liquid nutrient and substrate nutrients are converted toproduct-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which bacterial cell populations may be immobilized andfermentations performed in which liquid nutrient and substrate media areconverted to product-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which fungal cell populations may be immobilized andfermentations performed in which liquid nutrient and substrate nutrientsare converted to product-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which yeast cell populations may be immobilized andfermentations performed in which liquid nutrient and substrate nutrientsare converted to product-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which eukaryotic animal cell populations may be immobilizedand fermentations performed in which liquid nutrient and substratenutrients are converted to product-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which either prokaryotic or eukaryotic plant cellpopulations may be immobilized and fermentations performed in whichliquid nutrient and substrate nutrients are converted toproduct-containing output liquid streams.

It is a further object of the present invention to provide a method andapparatus by which enzymes or enzyme systems immobilized on solidsupports or catalysts immobilized on solid supports or cells or cellcomponents immobilized on solid supports may be immobilized andcatalyzed chemical conversions be effected in which liquid substratenutrients are converted to product-containing output liquid streams.

Another object of the present invention is to provide a method andapparatus by which dissolved oxygen concentrations (or other dissolvedgases) in the nutrient liquid flow directed into a bioreactor chambermay be raised to any desired level, depending on the applied hydraulicpressure.

Another object of the present invention is to provide a method andapparatus by which either a nutrient gaseous substrate (such as oxygen)in the nutrient input liquid flow directed into a bioreactor chamber oran excreted respiratory gas (such as, for example, carbon dioxide) inthe output liquid flow may be maintained in the dissolved state untilliquid-gas disengagement is desired, generally far downstream of thebioreactor chamber(s).

Another object of the present invention is to provide a method andapparatus by which the conversion of an available chemical substrateinto a desired product may be effected by a series of stepwisebiocatalyst-mediated conversions in which each chemical conversion stepis effected by one of a series of bioreactor chambers inserted seriallyor in parallel into the flow stream.

Another object of the present invention is to provide a non-specific,general method and apparatus for cell culture or fermentation which canbe applied to any cell type without significant variation.

It is yet another object of the present invention to provide a methodand apparatus by which biocatalysts are immobilized within bioreactorchambers while media containing toxic chemicals are fed into thebioreactor chambers and the biocatalysts in the bioreactor chambersneutralize the toxic chemicals thereby converting them into anenvironmentally benign products.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which significantly reducesboth the capital and labor costs of production and productionfacilities.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which is much lesssusceptible to contamination by opportunistic organisms.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation in which the liquidenvironment bathing the desired biocatalyst is essentially invariant intime, i.e., the pH, ionic strength, nutrient concentrations, wasteconcentrations, or temperature do not vary as a function of time in thebiocatalyst's environment.

Another object of the present invention is to provide a continuousfermentative or cell culture method.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation in which cycles ofproliferation, growth, or product formation can be accomplished simplyby varying the input nutrient feed composition.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which can continue for thelifetime(s) of the immobilized micro-organism or cell type.

Another object of the present invention is to provide a method andapparatus for culturing biocatalysts under conditions which therebysignificantly increases the yield of products from the biocatalyst.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which increases theconversion efficiency (of substrate to product) of the culture process.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which significantly reducesthe cost of heating or cooling the aqueous media required to support theculture process.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as antibiotics from micro-organismfermentations.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as enzymes or other proteins from micro-organismfermentations.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as ethanol or other short-chain alcohols andacids from the fermentation of micro-organisms.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as protein hormones from genetically-transformedmicro-organisms.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as protein hormones from eukaryotic cells.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as amino acids, nitrogenous bases, or alkaloidsfrom the fermentation of micro-organisms.

Another object of the present invention is to provide a method andapparatus for cell culture or fermentation which results in higheryields of products such as fuel-grade ethanol from the fermentation byyeasts of sugar-containing agricultural material.

Another object of the present invention is to provide a method andapparatus which would reduce the fermentation time required to producealcoholic beverages such as beer and wine.

Another object of the present invention is to provide an easilyscaled-up method and apparatus for cell culture or fermentation whichcan be commercially employed.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated and form a part of thespecification, illustrate several scientific principles and embodimentsof the present invention and, together with the description, serve toexplain the principles of the invention.

FIG. 1 illustrates the central features of Counter-Flow Centrifugation.

FIG. 2 illustrates an analysis of the operative forces in Counter-FlowCentrifugation.

FIG. 3 illustrates the central problem with Counter-Flow Centrifugation.

FIG. 4 illustrates the mathematical defect in the conventional treatmentof Counter-Flow Centrifugation.

FIG. 5 is an illustration of the effect on immobilized particles usingconventional Counter-Flow Centrifugation at long time periods.

FIG. 6 illustrates the modification of Counter-Flow Centrifugationemployed in the process of this invention.

FIG. 7 is an illustration of the mathematics governing the motion of aparticle due to the effect of gravity on that particle when it isrestrained in a centrifugal field exactly opposed by a liquid flow.

FIG. 8 is an illustration of the resultant motion of a particle underthe constraints of FIG. 7.

FIG. 9 is a mathematical evaluation of the immobilization conditions ata given radius.

FIG. 10 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating cylindrical bioreactor chamber.

FIG. 11 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating conical biocatalyst immobilizationchamber.

FIG. 12 is an illustration of a three-dimensional array of particles ina rotating conical biocatalyst immobilization chamber.

FIG. 13 is an illustration of the inter-stratum “buffer regions” in athree-dimensional array of particles in a rotating conical biocatalystimmobilization chamber.

FIG. 14 is a mathematical analysis of the intra-stratum flow velocityvariation in a two-dimensional array of particles in a rotating conicalbiocatalyst immobilization chamber.

FIG. 15 is an illustration of an example conical biocatalystimmobilization chamber and the boundary conditions which determine thosedimensions.

FIG. 16 is an analysis of the positional variation of the centrifugaland flow velocity forces in the chamber of FIG. 15. at a flow rate of 10mL/min.

FIG. 17 is a block diagram of a process configuration designed tomaintain desired dissolved gas concentrations in the liquid input to acentrifugal bioreactor.

FIG. 18 is an illustration of a representative liquid flow pressureregulator.

FIG. 19 is a sectional view of a first embodiment of the CentrifugalFermentation Process when viewed parallel to the axis of rotation.

FIG. 20 is a view of the rotor body of FIG. 19 when viewed parallel tothe axis of rotation.

FIG. 21. is a cross-sectional view of one of the demountable bioreactorchambers of FIG. 19.

FIG. 22 is a sectional view of the rotor body of FIG. 19 when viewedperpendicular to the axis of rotation.

FIG. 23 is a sectional view of the rotor body of FIG. 19 along thedotted line indicated in FIG. 22, when viewed parallel to the axis ofrotation.

FIG. 24 is a sectional view of the rotor body of FIG. 19 along thedotted line indicated in FIG. 22, when viewed parallel to the axis ofrotation.

FIG. 25 is a sectional view of the rotor body of FIG. 19 along thedotted line indicated in FIG. 22, when viewed parallel to the axis ofrotation.

FIG. 26 is a sectional view of the rotor body of FIG. 19 along thedotted line indicated in FIG. 22, when viewed parallel to the axis ofrotation.

FIG. 27 is a sectional view of the rotor body of FIG. 19 along thedotted line indicated in FIG. 22, when viewed parallel to the axis ofrotation.

FIG. 28 is an illustration of the axial channels and their termini inthe rotating shaft of FIG. 19.

FIG. 29 is a detail view of the distribution hub of the rotating shaftof FIG. 28.

FIG. 30 is a sectional view of a representative high-performance endface seal.

FIG. 31 is a sectional view of a second embodiment of the CentrifugalFermentation Process when viewed parallel to the axis of rotation.

FIG. 32 are views of the rotor body of FIG. 31 when viewed parallel tothe axis of rotation.

FIG. 33 is a cross-sectional view of one of the bioreactor chambers ofFIG. 31.

FIG. 34 is a sectional view of the rotor body of FIG. 31 when viewedperpendicular to the axis of rotation.

FIG. 35 is an illustration of the axial channels and their termini inthe rotating shaft of FIG. 31.

FIG. 36 is a sectional view of a representative high-performance endface seal.

FIG. 37 is a graphical and mathematical representation of the portion ofthe biocatalyst immobilization chamber of FIGS. 21 and 33 whichresembles a truncated cone.

FIG. 38 is a graph relating the flow rates and rotor speeds whichprovide for particle immobilization under the dimensional and boundarycondition constraints shown on FIG. 15 and for the rotor body of FIGS.19 and 31. for particles of sedimentation rates of 0.001 and 0.01 mm/minat flow rates up to 10 mL/min.

FIG. 39 is a graph relating the flow rates and rotor speeds whichprovide for particle immobilization under the dimensional and boundarycondition constraints shown on FIG. 15 and for the rotor body of FIGS.19 and 31 for particles of sedimentation rates of 0.1, 1.0, and 10.0mm/min at flow rates up to 10 mL/min.

FIG. 40 is a graph relating the flow rates and rotor speeds whichprovide for particle immobilization under the dimensional and boundarycondition constraints shown on FIG. 15 and for the rotor body of FIGS.19 and 31 for particles of sedimentation rates of 0.1, 1.0, and 10.0mm/min at flow rates up to 100 mL/min.

FIG. 41 is a graph displaying the relationship between rotor size andvolume capacity in a first embodiment of this invention.

FIG. 42 is a graph displaying the relationship between rotor size andvolume capacity in a second embodiment of this invention.

FIG. 43 is a graph displaying the relationship between rotor size androtational speed required to maintain a Relative Centrifugal Force of100×g in embodiments of the process of this invention.

FIG. 44 is a block diagram of a centrifugal process configurationdesigned to allow serial processing of a precursor chemical through twocentrifugal bioreactors.

FIG. 45 is an embodiment which may be employed for applications wherethe immobilized biocatalyst is in a complex consisting of a supportparticle to which the biocatalyst is attached.

FIG. 46 depicts the results of an example experiment in which, after ca.1×10¹⁰ P. putida cells were injected into and immobilized in the CBR, aflow of 5 ppm uranyl nitrate (pH=4.7) was started. The CBR output wasmonitored by ICP-AES for uranyl ion throughput.

FIG. 47 shows the time course of xylanase production from an A.pullulans culture initially grown up on glucose and subsequentlyswitched (at T=0) to xylose as the media carbon source.

FIG. 48 depicts the result of an analysis of the input vs. the outputlevels of nitrate ion as measured amperiometrically.

FIG. 49 shows one CBR embodiment to generate ethanol by, for example,anaerobic fermentation of glucose to ethanol by an immobilizedfermentative yeast population.

FIG. 50 shows one CBR embodiment to generate replacement microbial cellsfor periodic introduction into a parallel array of biocatalystimmobilization chambers.

DETAILED DESCRIPTION OF THE INVENTION

The development of this immobilization and culture process has itsorigin in four distinct areas of knowledge. The function of the overallprocess depends on the use of information from all four areas for itsproper function. These areas are: (1) Stoke's Law and the theory ofcounterflow centrifugation; (2) the geometrical relationships of flowvelocity and centrifugal field strength; (3) Henry's Law of Gases; and,(4) the effect of hydraulic pressure on single and multicellularorganisms and their cellular or subcellular components.

The central purpose of the process of this invention is immobilizationof three-dimensional arrays of particles (cells, subcellular structures,or aggregated biocatalysts) and to provide them with a liquidenvironment containing dissolved gases which will maximize theirviability and productivity. Such cells may include, but are not limitedto, a prokaryotic cell, a bacterium, or a eukaryotic cell, such as algaecells, plant cells, yeast cells, fungal cells, insect cells, reptilecells and mammalian cells. The biocatalyst may be, but is not limitedto, a subcellular component, an enzyme complex, and/or an enzyme compleximmobilized on a solid support.

The dissolved gases of the present invention include but are not limitedto air, O₂, NH₃, NO₂, Ar, He, N₂ and H₂ or any mixture thereof.

This process utilizes a novel modified form of “CounterflowCentrifugation” to immobilize particle arrays. A proper application ofStoke's Law in combination with provision for the effect of gravitywhich also acts on the immobilized particles results in a mathematicalrelationship which allows for the relative immobilization ofhigh-density arrays of such particles. The effect of gravity discussedpreviously and graphically depicted in FIGS. 3-5 can be eliminated by analternative choice of rotational axis as is shown in FIG. 6. If rotationabout the horizontal axis (y) is chosen instead of rotation about thevertical axis (z), as is most common in biological centrifugations, thenthe effect of gravity on immobilized particles will always be limited toaction solely in the x-z plane. Since this is the same plane in whichboth the centrifugal as well as the liquid flow related forces areconstrained to act, the motion of a restrained particle at any point ina rotational cycle is the resultant of the sum of the three types offorces acting upon it.

As is shown in Inset A of FIG. 7, where the plane of the Figure is thex-z plane, the effect of gravity (F_(g)) on the position of a particlesuspended in a radially-directed centrifugal field (F_(c)) while anexactly equal and opposing force supplied by an inwardly-directedflowing liquid (F_(b)) is directed toward the particle, can becalculated by the evaluation of equations 1-4 where (k) represents thedownward displacement in the x-z plane imparted by gravitational forcesduring an angular rotation of the rotor position equal to (a). Analysisof the motion of a particle under these constraints and for [2_X(k/a)]<R (a low mass particle) results in the determination that themotion is periodic; that is, the particle motion results in a return toits starting place after a complete rotation of 360 degrees (afterequilibrium is reached). As is shown in FIG. 7, the effect of gravity onthe motion of a particle otherwise immobile as a result of the opposingequality of the centrifugal and flow-related forces results in adecrease in radial position in quadrants I and II, and an exactly equalradial lengthening in quadrants III and IV. Thus, the radial distance ofthe particle from the axis of rotation also exhibits a periodic motionover the course of a full rotation of 360 degrees. It should be notedthat, mathematically, measurement of the periodicity of motion requiresonly one rotation if measurement begins at either 90 or 180 degreeswhereas two full rotations are required if measurement begins at eitherzero or 180 degrees, since a new equilibrium radial distance differentfrom the original results in the latter case.

The effective motion of a particle through a complete rotational cycleis shown in the inset of FIG. 8. If the sides of a container in whichthe particle is suspended are labeled 1 and 2 (see circled numbers inFIG. 8), then the motion of the particle over the course of onerotational cycle would describe a circle with its center displacedtoward the “leading edge” side of the particle's container. Thus, aparticle suspended in a centrifugal field which is opposed by an equalliquid flow field will be constrained to periodic motion (and thus iseffectively immobilized) if the balance of the radially-directed forcescan be maintained over the course of its movement.

With these theoretical considerations in mind, we can now return to thehypotheses of Sanderson and Bird which were graphically shown in FIG. 2.A corrected graphical representation is shown in FIG. 9, in which theaxis of rotation is now the (y) axis. Under these conditions thehypothesis of Sanderson and Bird can now be restated and applied tolong-term immobilization of particles. Equation 3 of FIG. 9 is nowvalid. There is a radial distance along the z axis (r_(z)) which, whenevaluated by Eqn. 3, represents a position in which the particle isrelatively immobilized in a centrifugal field which is exactly opposedby an inwardly-directed liquid flow, even in the presence of agravitational field. Furthermore, a simplification of Stoke's Law(Eqn. 1) under the conditions of uniform particle size, shape, anddensity and a homogeneous liquid flow results in Eqn. 2, where it isobvious that the Sedimentation Velocity of a particle (SV) is a simplelinear function of the applied centrifugal field. Similarly, Eqn. 3 canthen be rewritten under the same conditions to yield Eqn. 4, whereliquid Velocity (V in Eqn. 3) has been replaced by liquid Flow Velocity(FV). Equation 4 suggests that there is a continuum of liquid flowvelocities and applied centrifugal fields which could be matched by theevaluation of constant (C), all of which would satisfy the requirementof relative particle immobilization. Further, if the liquid flowvelocity could be varied as a function of (z), there could be a separateapplication of this equation at each radial distance. Consideration ofthe implications of Eqn. 4 is important for the relative immobilizationof three-dimensional arrays of particles as opposed to theimmobilization of two-dimensional arrays of particles at a single radialdistance from the rotational axis.

If the biocatalyst immobilization chamber in which a particle is locatedis cylindrical (as is graphically depicted in FIG. 10) and if a liquidis flowed into this chamber from the end of the chamber most distal tothe axis of rotation, then it is obvious that the flow velocity of thisliquid flow (as defined in Eqn. 1, FIG. 10) will have a single value atall points not occupied by layers of particles. As a consequence, if atwo-dimensional array of particles is in positional equilibrium at aparticular radial distance (A₁), as is indicated in Eqn. 2, (where CF isthe centrifugal field strength and FV is the liquid flow velocity) thenparticles forced to occupy positions at radial distances either greaterthan or smaller than A₁, such as those located in FIG. 10 at A₂ or A₃,will necessarily be presented with an inequality of restraining forceswhich will result in net translation of the particles. Thus, thoseparticles located at A₂, a longer radial distance than A₁, willexperience a greater centrifugal force than those at A₁ and willnecessarily migrate to longer radial distances (Eqn. 3). Conversely,particles initially located at A₃ would experience a reduced centrifugalfield and would migrate to shorter radial distances (Eqn. 4). Thus, itis not possible to form a three-dimensional array of particles in a“parallel-walled” biocatalyst immobilization chamber such as that ofFIG. 10.

If, however, the biocatalyst immobilization chamber has a geometry suchthat its cross-sectional area increases as the rotational radiusdecreases, as is graphically displayed in FIG. 11, then it ismathematically possible to form three-dimensional arrays of immobilizedparticles. This is a consequence of the fact that the microscopic flowvelocity of the liquid flow varies inversely as the cross-sectional area(Eqn. 1) while the relative centrifugal field varies directly as therotational radius (Eqn. 2). Thus, if values of flow velocity androtation velocity are chosen such that a two-dimensional array ofparticles is immobilized at rotational radius A₁ (Eqn. 3), then it ismathematically possible to adjust the “aspect ratio” of the side wallsof the biocatalyst immobilization chamber such that those particlesinitially located at radial distance A₂ could also experience either ansimilar equality of forces or, as is shown in Eqn. 4, an inequality offorces which results in net motion back toward the center of thechamber. A similar argument may be applied to particles located at A₃(see Eqn. 5). Although the geometry of the biocatalyst immobilizationchamber as depicted in FIG. 11 is that of a truncated cone, note thatother geometries could be alternatively used—subject to the constraintthat the cross-sectional area of the chamber increases as the rotationalradius decreases. Thus, as is depicted in FIG. 12, it is possible toconstruct a three-dimensional array of particles in a varyingcentrifugal field opposed by a liquid flow field if the biocatalystimmobilization chamber geometry chosen allows for a flow velocitydecrease greater than or equal to the centrifugal field strengthdecrease as the rotational radius decreases. In the geometry chosen inFIG. 12, that of a truncated cone, the two-dimensional arrays ofparticles at each rotational radius (R_(c)) will each be constrained tomotion toward that radius where the opposing forces are exactly equal.

While, at first glance, the description presented above would suggestthat the net effect of the mismatch of forces at all radii other thanthat which provides immobilization would result in a “cramming” of allparticles into a narrow zone centered on the appropriate radius, such isnot the case. As is shown graphically in FIG. 13, as each layer ofparticles approaches an adjacent layer, it will move into a region wherea “cushioning effect” will keep each layer apart (the horizontal arrowsin FIG. 13). The explanation for the inability of adjacent layers ofparticles to interdigitate is a consequence of an analysis of themicroscopic flow velocity profile through each layer. In FIG. 14, asingle representative stratum of spherical particles confined to aparticular radial distance in a chamber layer of circular cross-sectionis presented. The ratio of the diameters of the particles to thediameter of the cross-section of FIG. 14 is 12:1. While the magnitude ofthe flow velocity of the liquid through unoccupied portions of thechamber cross-section can be quantified simply from the chamberdimensions at that point, the flow velocity through a region occupied bya stratum of particles will necessarily be much greater than that in theabsence of a stratum of particles because of the greatly reducedcross-sectional area through which the liquid must travel. As is shownin the graph in FIG. 14, the increase in flow velocity through a stratumof the above dimensions is more than double that determined in the freespace just adjacent to the stratum on each side. This microscopicincrease in local flow velocity in the region of each stratumeffectively provides a “cushion” which keeps each adjacent stratumseparate.

In actual use, it has been determined that, for the case of a chambergeometry of a truncated cone, it is preferable that the most distalregion of the truncated cone be the region where an exact equality ofcentrifugal forces and liquid flow velocity is achieved. The “aspectratio” (the ratio of the small radius of the truncated cone to the largeradius of the truncated cone) of the truncated cone is determined by thesimultaneous solution of the two equations presented in FIG. 15. In Eqn.2, the desired boundary condition of immobility for that “lowest”stratum of particles is presented. It states that the intrinsicsedimentation rate of the particle due to gravity (SR) times therelative centrifugal field applied at that radial distance (RCF) beexactly equal to the magnitude of the liquid flow velocity (FV) at thatpoint. In Eqn. 1, a desired boundary condition at the opposite surfaceof the array of particles is presented. In order to insure retention ofall particles within the biocatalyst immobilization chamber, a boundarycondition wherein the product of SR and RCF is twice the magnitude ofthe flow velocity at that radial distance has been arbitrarily chosen.Simultaneous solution of the desired boundary condition equations isused to solve for the ratio of the conic section diameters when theupper diameter and conic length is known.

FIG. 16 is a profile of the relative magnitudes of the flow-relatedforces and the centrifugal forces across a biocatalyst immobilizationchamber of conical cross-section which has dimensions in this exampleof: large diameter=6.0 cm, small diameter=3.67 cm, and depth=3.0 cm. Wedefine the Relative Sedimentation Rate as the product of the intrinsicsedimentation rate of a particle due to gravity in a nutrient media atits optimal temperature and the applied centrifugal field. For a givenflow rate (in this example 10 mL/min) into a biocatalyst immobilizationchamber of the indicated dimensions, where the proximal end of thebiocatalyst immobilization chamber is 9.0 cm from the rotational axis,the product of the intrinsic particle sedimentation rate due to gravityand the angular velocity is a constant at the given flow rate in orderto satisfy the desired boundary conditions (see FIG. 15). In otherwords, the angular velocity need not be specified here since its valuedepends only on the particular particle type to be immobilized. Thedotted line in FIG. 16 displays the linear variation in the centrifugalfield strength from the bottom to the top of the biocatalystimmobilization chamber, while the solid line displays the correspondingvalue of the flow velocity. At the bottom of the chamber (the mostdistal portion of the chamber), the forces are equal and a particle atthis position would experience no net force. At the top of the chamber,a particle would experience a flow-related force which is only one-halfof the magnitude of the centrifugal field and would thus be unlikely toexit the chamber, even in the presence of a nearby region of decreasingcross-sectional area (the chamber liquid exit port), where flowvelocities will increase markedly.

It should be clear from the foregoing that, subject to the necessarycondition that the cross-sectional area increases as rotational radiusdecreases, there are other geometrical chamber configurations whoseshape could be manipulated in order to establish boundary andintermediate relationships between the applied centrifugal field and theliquid flow velocity forces at any radial distance in order to establishdesired resultant force relationships in the three-dimensional particlearrays. In practice, however, it is undesirable to utilize geometrieswith rectangular cross-sections as a result of the anomalous effects ofcoriolis forces which act in a plane transverse to the rotational plane.In the case of rectangular cross-sections, these otherwise unimportantforces can contribute to interlayer particle motion.

It should also be clear from the foregoing that the effect ofgravitational forces acting on the individual particle masses which actsindependently of the applied centrifugal forces (see FIGS. 7-8) are evenless important than was indicated earlier. In particular, since thebasic effect of gravity on an otherwise immobilized particle is toeither cause radial lengthening or radial shortening, such a motion of aparticle will necessarily bring it either into a region of increasedflow velocity magnitude (longer radii) or decreased flow velocitymagnitude (shorter radii) with only a much smaller change in centrifugalfield strength (see FIG. 16).

As a consequence, the periodic motion of a particle due to gravitationaleffects on its intrinsic mass will be severely dampened in the presenceof such unbalanced opposing force fields and will amount to, in the caseof low mass particles such as biocatalysts, a “vibration in place.”

It should also be obvious from the foregoing that there could be, in apractical sense, a severe problem with the maintenance of theimmobilized particle arrays in the above fashion when these particlesare aerobic cells, micro-organisms, or biocatalytic substructures. Suchstructures require, in addition to liquid nutrients, the provision ofcertain nutrients which are gases at ambient temperatures and pressures.For example, the large majority of cells or micro-organisms which arevaluable in the production of commercial biochemicals are aerobes. Thatis, they require oxygen for viability. While these living organisms (ortheir subcellular constituents) can only utilize oxygen in a dissolvedform, the only method of providing oxygen heretofore was by bubbling orsparging oxygen through the nutrient liquid in which the cells aresuspended in order to effect the solubilization of oxygen. Further, mostliving organisms (including certain anaerobes) produce metabolic wasteswhich are gases (for example, carbon dioxide or methane). If gas volumeswere either introduced into or generated from metabolic processesoccurring in the immobilized three-dimensional arrays of particlesdiscussed above, then the careful balance of forces which provides fortheir immobilization would be destroyed.

Thus, the proper function of the centrifugal immobilization process ofthis invention requires that provisions be made to eliminate thepossibility of either the introduction of, or the generation of, gas(es)within the biocatalyst immobilization chamber. Since the only form ofthese otherwise gaseous chemicals which is utilizable by these cells (oris produced by them) is the aqueous dissolved form, it is this formwhich must be preserved in the process of this invention. One may ensurethis condition by the application of Henry's Law, which, in essence,states that the quantity of a gas which may be dissolved in a liquid isa function of the system pressure. Thus, if the hydraulic pressure ofthe liquid-containing system (the biocatalyst immobilization chamber andthe liquid lines leading to and from the biocatalyst immobilizationchamber) are maintained at a hydraulic pressure sufficient to fullydissolve the necessary quantity of input gas and to insure thesolubility of any produced gases, then there will be no disturbance ofthe immobilization dynamics.

As used herein, the terms “biocatalyst immobilization chamber”, “reactorchamber”, “bioreactor chamber”, “cell confinement chamber”, “centrifugalconfinement chamber”, “centrifugal cell chamber”, “immobilizationchamber”, “chamber”, “compartment”, or “confinement chamber” are allequivalent descriptive terms for the portion of the invention describedherein where cells or biocatalysts are suspended by the describedforces. Use of these equivalent terms does not imply an estoppel orlimitation of the description of the invention.

FIG. 17 is a block diagram which demonstrates one method by which themaintenance of such a gas-free, completely liquid system at hydraulicpressures greater than ambient may be effected. In this system, theindicated pumps are all positive displacement pumps. That is, liquid isconstrained to motion through the pumps in the directions indicated bythe arrows. Pump 3 is the primary feed pump which moves liquids into andout of the cell immobilization chamber which is located in a centrifugerotor. The raising of the hydraulic pressure in the circuit containingPump 3 and the cell immobilization chamber is accomplished by placing aliquid pressure regulator, the system pressure regulator, at a positionin the circuit downstream of the cell immobilization chamber. Thus, thesetting of a pressure limit higher than ambient on the system pressureregulator results in no liquid flow through this circuit until thepositive displacement pump, Pump 3, moves enough liquid into the circuitto raise the system hydraulic pressure to a value near this setting.Once an equilibrium system pressure is established, the pressurizedliquid downstream of Pump 3 will flow continuously at a rate set bycontrol of Pump 3.

In order to dissolve an appropriate amount of a desired nutrient gasinto the liquid input to Pump 3, a Gas-Liquid Adsorption Reservoir isplaced in the input line leading to Pump 3. Non-gassed liquids are movedfrom the Media Reservoir into the Gas-Liquid Adsorption Reservoir bymeans of Pump 1. Quantities of the desired gas (air or oxygen, forexample) are, at the same time, let into the Gas-Liquid AdsorptionReservoir through a pressure regulator set for the gas pressure requiredto insure the solubilization of the desired concentration of the gasinto the nutrient liquid. Note that, in the steady-state, it isnecessary that Pump 1 be operated at the same flow rate set for Pump 3.Pump 2 is a recirculation pump which is operated at a flow rate higherthan that of Pumps 1 and 3. Pump 2 is used to increase the contactbetween the gas and liquid phases of the Gas-Liquid Adsorption Reservoirso that a desired concentration of gas dissolved in the nutrient liquidis maintained in the bulk of the volume of liquid in the Gas-LiquidAdsorption Reservoir. It is essential, because of the nature of positivedisplacement pumps, that the magnitude of the system pressure set withthe System Pressure Regulator be higher than the pressure magnitude setin the Gas-Liquid Adsorption Reservoir. In order to make available, atany time, a sufficient volume of liquid equilibrated with the desiredconcentration of gas(es), a valve on the input to Pump 3 may be utilizedto allow such equilibration to occur prior to any actual use. Similarly,by means of switching valves, the liquid input to Pump 3 may be changedfrom that indicated in FIG. 17 to any other input reservoir desired,subject to the constraint that the hydraulic pressure of such areservoir be lower than the value of hydraulic pressure set by theSystem Pressure Regulator.

FIG. 18 is a depiction of a representative, commercially-availableliquid pressure regulator. A flow of liquid 14 into the pressureregulator is obstructed by a spring-loaded needle valve 10 which pressesagainst a seat 11. When the hydraulic pressure of the input liquidbecomes great enough, the needle valve 10 is displaced from the seat 11and a flow can then exit (as indicated by line 15) the pressureregulator. The fixed pressure exerted by the needle valve spring 12 canbe adjusted by increasing or decreasing the pressure exerted by theadjustable spring 13.

It should be obvious that the block diagram of FIG. 17 is arepresentation of one of many process flow configurations which may beemployed in order to flow a gas-free pressurized liquid through acentrifugal bioreactor chamber. In particular, one may envision manydifferent methods of insuring adequate mixing of gas and liquid in orderto effect the solubilization of a measured quantity of gas into theliquid. What is central to the process of this invention is: (1) thatthe liquid circuit comprising the bioreactor chamber and the liquidtransport lines (into and out of the bioreactor chamber) be operated ata hydraulic pressure greater than ambient pressure; (2) that there beprovision for the solubilization of a desired quantity of a gas into theliquid prior to its insertion into the liquid circuit leading to thebioreactor chamber(s); and (3) that the system hydraulic pressure bemaintained at a high enough value to keep both the input gas(es), aswell as the respiratory gas(es) which may be produced by biologicalsystems in solution throughout the liquid circuit, upstream of thesystem pressure regulator and downstream of Pump 3. Hydraulic pressuresof 100-2000 psig have proved sufficient to maintain a gas-free liquidenvironment for all possible conditions of cell density and cell number.

There will be no measurable deleterious effects on the culture of animalcells or micro-organisms or their subcellular constituents as a resultof the necessity to increase the hydraulic pressure of their environmentin the biocatalyst immobilization chamber at hydraulic pressures below10,000 psig. The successful culture of living cells using bioreactorheadspace pressurization is a proven and established culture method,albeit limited in scope to pressures of less than 50 psig (see Yang, J.and Wang, N. S. (1992) Biotechnol. Prog. 8, 244-251 and referencestherein). At hydraulic pressures of 15,000 to 30,000 psig somedisassociation of noncovalent protein complexes has been observed,although pressures of more than 90,000 psig are required to denaturemonomeric proteins (Yarmush, et al. (1992) Biotechnol. Prog. 8,168-178). It is a seldom appreciated, but well known fact that livingcells (and their constituent parts) are unaffected by, and indeed cannotsense hydraulic pressure magnitudes below those limits outlined above.This may best be appreciated in considering the effects of hydraulicpressure on marine organisms. For every 10 meters of depth under thesea, approximately one atmosphere (14.7 psig) of overpressure is gained.Thus, for example, benthic organisms exhibiting biochemical processesand metabolic pathways identical to their shallow-water and terrestrialcounterparts inhabit ecological niches and proliferate mightily athydraulic pressures of more than 3000 pounds per square inch. Similarly,the hydraulic pressure under which terrestrial mammalian cells exist isgreater than ambient, ranging from ca. 90 to 120 mm Hg greater thanambient in man, for example. The explanation for the “invisibility” ofhydraulic pressure in biological systems can be understood if it isrealized that hydraulic pressure in aqueous systems has, as its “forcecarrier,” the water molecule. Since the lipid bilayer which forms theboundary membrane of living cells is completely permeable to watermolecules, an applied hydraulic pressure in aqueous systems istransmitted across the boundary membranes of cells or subcellularorganelles by the movement of water molecules with the result that theinterior(s) of cells rapidly equilibrate to an externally-appliedaqueous hydraulic pressure.

There are situations in which hydraulic pressures are deleterious toliving cells. For example, if a pressure field in an aqueous system isvaried at high frequency, then it is possible to cause cell disruptionby means of pressure differentials across the cell boundary membrane.However, the frequency required for such lethal effects is quite high;on the order of thousands of cycles per second. As long as the pulsatilepressure of pumping in the process of this invention is kept below sucha limit there is no effect on cell viability for even the most fragileof cells as a result of pressure fluctuations. In addition, cellreplication is completely unaffected by culture at increased hydraulicpressure.

The problem of the introduction and withdrawal of pressurized liquidflows into and out of a rotating system has been solved by innovationsin seal design over the past twenty years. High performance mechanicalend-face seals are available which are capable of operation atrotational rates in excess of 5000 revolutions per minute whilemaintaining a product stream hydraulic pressure of more than 2000 psig.Such seals are available from Durametallic Corporation (2104 FactoryStreet, Kalamazoo, Mich. 49001). Such high-performance mechanical sealshave leakage rates below 5 liters per year, can be cooled by pressurizedrefrigerated liquids of which inadvertent leakage into the productstream at the above leakage rates will have no effect on biologicalsystems, and can be operated in a manner which provides for themaintenance of absolute sterility in the product stream. The somewhatinexplicable aversion to the use of mechanical end-face seals for use incentrifugal bioreactor systems (see U.S. Pat. Nos. 4,939,087 and5,151,368, for example) results in a perceived necessity for theconnection of flexible tubing (and complicated mechanisms for its“untwisting”) in conventional designs. Such designs are, as a result,limited to: (1) hydraulic pressures near one atmosphere as a consequenceof tube flexibility requirements; and (2) low rotational speeds andshort bioreactor run times as a result of the vigorous motion of theseflexing connections. The use of modern high performance mechanicalend-face seals eliminate all of these drawbacks to centrifugalbioreactor performance.

Immobilization of three-dimensional arrays of particles in a forcefield, which is comprised of outwardly-directed centrifugal forces whichare opposed by inwardly-directed liquid flow forces has been described.The effect of gravitational forces which act, inevitably, on even thesmallest and lightest of particles over prolonged time periods can beessentially negated and reduced to a small periodic “vibration in place”by the proper choice of rotational axis. The disruptive effects of thepossible introduction of gases into this system have been accounted forby raising the hydraulic pressure of the liquid system to values whichassure that such otherwise gaseous chemicals will remain dissolved inthe flowing liquid. It has been emphasized that the necessary increasein hydraulic pressure will have no effect on biological units such ascells, microorganisms, or their subcellular constituents.

In the following paragraphs, we present and analyze two embodiments ofthe invention. FIG. 19 depicts the components of a first embodiment ofthe present invention. A cylindrical rotor body 20 is mounted on ahorizontal, motor-driven rotating shaft 21 inside a safety containmentchamber 22 bounded by metal walls. The rotor body 20 is fixed inposition on the rotating shaft 21 by means of locking collars 23. Therotating shaft 21 is supported on either side of the rotor body 20 bybearings 24. The rotating shaft 21 extends outside the safetycontainment chamber 22 for a distance and ends in a terminal bearing andend cap 29 mounted in an external housing 25. Liquid flows areintroduced into and removed from bioreactor chambers 26 mounted in therotor body 20 by means of a liquid input mechanical end-face seal 28 anda liquid output mechanical end-face seal 27 which communicate withliquid channels (50, 51 in FIG. 22) within the rotating shaft 21.Typical dimensions for an example rotor body 20 (a=36 cm and b=15 cm)are entirely reasonable and comparable to rotor dimensions known tothose skilled in the art.

FIG. 20 is a view of the rotor body 20 of FIG. 19 as viewed parallel tothe axis of rotation. The rotor body 20 is machined with a shaftmounting channel 30 through its center to allow its mounting on therotating shaft (21 in FIG. 19), and is machined to havechamber-positioning recesses 32 into which cylindrical demountablebioreactor chambers (26 in FIG. 19) may be placed. The rotor body 20 isalso machined to have radial rectilinear channels 33 (such as thecentrally-located axial liquid output channel 51 in FIG. 22, and theeccentric axial liquid input channel 50 in FIG. 22) in which liquidlines (such as the output liquid transport lines 53 in FIG. 22 and theinput liquid transport lines 54 in FIG. 22) which communicate with thebioreactor chambers (26 in FIG. 22) may be located. In actual use, acircular cover (not shown) would be attached to the surface of the rotorbody 20 to close the rotor body 20.

FIG. 21 is a depiction of one of the bioreactor chambers 26 of FIG. 19.The bioreactor chamber (26 in FIG. 19) is cylindrical and is composed oftwo pieces of thick-walled metal; a top piece 40 and a bottom piece 42.The top piece 40 contains a machined conical recess 47 and a machinedpassage 48 terminating in an output compression fitting 41 by whichliquid may be removed from the bioreactor chamber (26 in FIG. 19). Thebottom piece 42 is made of the same metal as the top piece 40, and isinternally machined to form a biocatalyst immobilization chamber 43 of adesired geometric shape. The shape of the biocatalyst immobilizationchamber 43 depicted in FIG. 21 is that of a truncated cone with a shortcylindrical volume at its top face and a short conical volume at itsbottom face. A machined passage 48 terminating in an input compressionfitting 44 allows liquid input into the biocatalyst immobilizationchamber 43. The top piece 40 and the bottom piece 42 of the biocatalystimmobilization chamber 43 are bolted together by means of countersunkassembly screws 45 and sealed against an internal positive hydraulicpressure by means of one or more O-ring compression seals 46. In thecase of certain animal cell cultures in which contact between theimmobilized cells and the interior metal walls of the biocatalystimmobilization chamber 43 should be avoided, it may be expedient toprovide suitable conical inserts of, for example, polyethylene, in orderto prevent such contact. Alternatively, the interior of the biocatalystimmobilization chamber 43 might be coated with an appropriate liningmaterial to provide the same effect.

FIG. 22 is a transverse sectional view through the rotor body 20 of FIG.19 parallel to the axis of rotation. The bioreactor chambers 26 areconnected to an eccentric axial liquid input channel 50 and to acentrally-located axial liquid output channel 51 within the rotatingshaft 21 by means of output liquid transport lines 53 and input liquidtransport lines 54. The output liquid transport lines 53 are metal tubeswhich communicate with the bioreactor chambers 26 and thecentrally-located axial liquid output channel 51 through outputcompression fittings 41. The input liquid transport lines 54 are metaltubes which communicate with the bioreactor chambers 26 and theeccentric axial liquid input channel 50 through input compressionfittings 44. The exact machining of the rotor body 20 may be examined byfive different sectional views of the rotor body 20 perpendicular to theaxis of rotation (see FIGS. 23-27) which are sectional views at thelevels indicated by the dotted lines in FIG. 22.

In FIGS. 23-27, the dimensions and configuration of five differentinternally-machined sections of the rotor body 20 of FIG. 19 aredisplayed. FIGS. 23 and 27 show one method by which the rotor body 20may be mounted on the rotating shaft (21 in FIG. 19) by means ofsprocket-shaped recesses 60 concentric with the shaft mounting channel30 which accept the locking collars (23 in FIG. 19). S-1 in FIGS. 23 and27 is a cross-sectional view of the shaft mounting channel 30 and thesprocket-shaped recesses 60. FIG. 24 depicts four radial rectilinearchannels 33 machined into the rotor body 20 into which the output andinput liquid transport lines (53 and 54, respectively, in FIG. 22) willtravel. FIG. 25 depicts the shapes of the chamber-positioning recesses32 machined into the rotor body 20 into which the bioreactor chambers(26 in FIG. 19) are placed, and also shows the relationship of thesechamber-positioning recesses 32 to the radial rectilinear channels 33.Note that the radial rectilinear channels 33 extend farther radiallythan do the chamber-positioning recesses 32 and thus provide a supportchannel against which the output and input liquid transport lines (53and 54, respectively, in FIG. 22) rest as they extend “upward” toconnect with an input compression fitting (44 in FIG. 21) of thebioreactor chambers (26 in FIG. 22). Because each input liquid transportline (54 in FIG. 22) is supported by resting against a wall of the mostdistal radial rectilinear channel 33 as the most distal radialrectilinear channel 33 makes a right angle bend to travel to itsterminus at an input compression fitting (44 in FIG. 21) of eachbioreactor chamber (see section S-2, FIG. 24), there is no extracentrifugal stress applied to the input liquid transport lines (54 inFIG. 22) as a result of the rotational movement of the system.

FIG. 26 details the internal machining of the rotor body 20 of FIG. 19for the liquid output line attachment recesses 70 necessary to provideworking room for the mechanical attachment of the output liquidtransport lines (53 in FIG. 22) to the bioreactor chambers (26 in FIG.19), using output compression fittings (41 in FIG. 21). As is shown inFIG. 22, the output liquid transport lines 53 are bent into a “U-shaped”configuration (exaggerated in FIG. 22) which allows their length to beadjusted during mechanical connection to the bioreactor chambers (26 inFIG. 19). The bioreactor chambers (26 in FIG. 19) are supported againstcentrifugal stress by the distal walls of the chamber-positioningrecesses 32; no weight is imparted to the output liquid transport lines(53 in FIG. 22) (except their own) as a result of centrifugal forces.

FIG. 28 is a view of the portion of the rotating shaft 21 on which therotor body 20 is mounted, and the portion of the rotating shaft 21 onwhich the liquid output mechanical end-face seal 27 and the liquid inputmechanical end-face seal 28, which convey liquid flows into and out ofthe bioreactor chambers 26, are mounted. The rotating shaft 21 containstwo axial liquid transport channels; the eccentric axial liquid inputchannel 50, and the centrally-located axial liquid output channel 51.The centrally-located axial liquid output channel 51 transports theliquid output of the bioreactor chambers 26 to the liquid outputmechanical end-face seal 27 by means of a short radially-directedconnecting passage 82 while the eccentric axial liquid input channel 50conveys liquid from the liquid input mechanical end-face seal 28 to thebioreactor chambers 26, also by means of a short radially-directedconnecting passage 81. The eccentric axial liquid input channel 50 andthe centrally-located axial liquid output channel 51 extend from one endof the rotating shaft 21 to the region where the rotor body 20 islocated. Compression plugs 80 seal the terminal axial openings of boththe eccentric axial liquid input channel 50 and the centrally-locatedaxial liquid output channel 51.

FIG. 29 is a view of the radially-disposed liquid distribution channelhubs in the region of the rotating shaft 21 where the rotor body (20 inFIG. 19) will be mounted. Two pairs of channels; the radial outputliquid line channels 90 and the radial input liquid line channels 92 aremachined through two cross-sections of the rotating shaft 21. The radialoutput liquid line channels 90 are in direct communication with theeccentric axial liquid input channel 50. In the case of the radial inputliquid line channels 92, an additional radial passage 94 is machinedwhich connects the eccentric axial liquid input channel 50 with thecentral connection of the radial input liquid line channels 92. Thisadditional radial passage 94 is sealed with a compression plug 95 at thesurface of the rotating shaft 21. In actual practice, particularly inthe case of high-speed operation of the invention, it may be preferablethat the eccentric axial liquid input channel 50, and thecentrally-located axial liquid output channel 51, be eccentric to theaxis of rotation and located symmetrically on a diameter of the rotatingshaft 21 for balancing purposes.

FIG. 30 is a view of a liquid output mechanical end-face seal assembly,such as the liquid output mechanical end-face seal 27, shown in FIG. 19.The liquid output mechanical end-face seal 27 is mounted on the rotatingshaft 21 and positioned with an opening to the interior liquid space ofthe seal over a short radially-directed passage 82 which communicateswith the centrally-located axial liquid output channel 51 machined intothe rotating shaft 21. A seal between the rotating and stationaryportions of the liquid output mechanical end-face seal 27 is provided bythe contact of the stationary seal face 100 against the rotating sealface 102. In the case of high performance end-face seals utilizable inthe process of this invention, where consideration must be made for theresultant centrifugal forces which act on the seal components, allspring elements are located in the stationary portion of the sealassembly. While the seal configuration shown in FIG. 30 is that of asingle seal, double and/or tandem end-face seal configurations may provemore advantageous in prolonged usage. Not shown in the figure arepressurized cooling liquid passages and jacketing necessary to maintaintemperature equilibrium in the seal assembly. When such a liquid outputmechanical end-face seal assembly is mounted on the rotating shaft 21 ofthe present invention, aqueous liquids may be pumped into the stationarypart of the seal assembly via compression fitting attachment, and thepumped liquid will follow the path indicated by the dotted line 103 tomake communication with the centrally-located axial liquid outputchannel 51 which transports this liquid away from the bioreactorchambers (26 in FIG. 28) mounted in the rotor body (20 in FIG. 28).

The principal disadvantage heretofore in the employment of mechanicalseals for the transfer of liquids into and out of rotating systems,where the purpose of the system is to culture biological entities suchas animal cells or micro-organisms, has been the problem of themaintenance of sterility. Low pressure mechanical seals have, in thepast, provided a route by which adventitious micro-organisms can gainentrance into bioreactor systems via the thin film of internal liquidwhich lubricates the end-face seal surfaces. In the process of thisinvention, where the internal liquid is always held at a hydraulicpressure higher than ambient, all leakage of liquid will occur to theexterior of the system. There is thus no possible route through whichadventitious contaminants can enter the system. Furthermore, the smallleakage of internal nutrient or product liquid which might exit thebioreactor system through the mechanical seals of the process of thisinvention (which might, for example, contain micro-organisms in certainapplications) will not be free to dissipate into the environment. As aconsequence of the operating characteristics of high-speed,high-pressure mechanical seals, it will be necessary to surround theseal components with a pressurized cooling liquid flow. It has beenfound, in practice, that an ideal liquid which possesses the properviscosity and flow properties for the cooling of such seals is 75-85%glycerol. Any leakage of internal liquid to the exterior in the processof this invention will result in its dispersion into the body of thisrecirculated liquid. We have found that glycerol at this concentrationis completely unable to support the growth of a number of representativeanimal cells or micro-organisms; this is likely a general phenomenon,presumably as a result of the osmotic movement of water out of theliving cells into the glycerol. Thus, periodic sanitary disposal of thecooling liquid volume of glycerol when it becomes diluted with leakagevolumes and its replacement with fresh glycerol will serve to maintainsterility in the single place in the system where liquids might escape.Finally, since it is possible, after prolonged use, that loss ofinternal system pressure or incipient failure of the seal systems mightallow liquid flow in the reverse direction across the seal faces, it isimportant to note that small quantities of glycerol which could thusleak into the bioreactor system would not be anything but an additionalnutrient when diluted into the flowing internal process liquid.

In order to obtain data for an analysis of the performance of a rotorbody of the dimensions and configuration outlined in FIGS. 19-20 and22-29 and containing demountable cylindrical bioreactor chambers (26 inFIG. 21), it was necessary that several scale dimensions and boundaryequations be chosen arbitrarily and used to determine the operatingcharacteristics of the first embodiment of the present invention. Theimmobilization boundary equations chosen are those listed in Equations 1and 2 of FIG. 15. The rotor dimensions chosen for this example andindicated by letter in FIGS. 19-29 are as follows:

a: 15.0 cm  j: 10.0 cm  s: 2.54 cm  b: 36.0 cm  k: 1.50 cm  t: 4.0 cm c:1.27 cm  l: 6.0 cm u: 6.14 cm  d: 1.0 cm m: 0.5 cm v: 1.0 cm e: 1.73 cm n: 1.0 cm w: 1.0 cm f: 3.0 cm o: 1.0 cm x: 6.5 cm g: 7.0 cm p: 5.0 cm y:5.0 cm h: 2.0 cm q: 6.0 cm z: 5.5 cm i: 2.0 cm r: 4.0 cm

FIG. 31 depicts the components of a second embodiment of this invention.A cylindrical rotor body 20 is mounted on a horizontal, motor-drivenrotating shaft 21 inside a safety containment chamber 22 bounded bymetal walls. The rotor body 20 is fixed in position on the rotatingshaft 21 by means of locking collars 23. The rotating shaft 21 issupported on either side of the rotor body 20 by bearings 24. Therotating shaft 21 extends outside the safety containment chamber 22 fora distance. Liquid flows are introduced into and removed from bioreactorchambers 26 in the rotor body 20 by means of a liquid input mechanicalend-face seal 28 and a liquid output mechanical end-face seal 27. Theliquid input mechanical end-face seal 28 communicates with acentrally-located axial liquid input channel (52 in FIG. 34) within therotating shaft 21. The liquid output mechanical end-face seal 27communicates with a centrally-located axial liquid output channel (51 inFIG. 34) within the rotating shaft 21. Typical dimensions for an examplerotor body 20 (a=10 cm and b=36 cm) are entirely reasonable andcomparable to rotor body dimensions known to those skilled in the art.

FIG. 32 shows two views of the rotor body 20 of FIG. 31. The rotor body20 is machined with a shaft mounting channel 30 through its center toallow its mounting on the rotating shaft (21 in FIG. 31) and is machinedto have mounting recesses 31 into which three rectangularly-faceddemountable bioreactor chambers may be placed.

FIG. 33 is a depiction of one of the bioreactor chambers of FIG. 31 (26in FIG. 31). The bioreactor chamber (26 in FIG. 31) is rectilinear insection and is composed of a top piece 40 and a bottom piece 42 ofthick-walled metal. The top piece 40 contains a machined conical recess47 and a machined passage 48 terminating in an output compressionfitting 41 by which liquid may be removed from the bioreactor chamber(26 in FIG. 31). The bottom piece 42 is made from the same metal as thetop piece 40 and has been internally machined to form a biocatalystimmobilization chamber 43 of a desired geometric shape. The shape of thebiocatalyst immobilization chamber 43 is that of a truncated cone with ashort cylindrical volume at its top face and a short conical volume atits bottom face. A machined passage 48 terminating in an inputcompression fitting 44 allows liquid input into the biocatalystimmobilization chamber 43. The top piece 40 and the bottom piece 42 ofthe biocatalyst immobilization chamber 43 are bolted together by meansof countersunk assembly screws 45 and sealed against an internalpositive hydraulic pressure by means of one or more o-ring compressionseals 46. In the case of certain animal cell cultures where contactbetween the immobilized cells and the interior metal walls of thebiocatalyst immobilization chamber 43 should be avoided, it may beexpedient to provide suitable conical inserts of, for example,polyethylene, in order to prevent such contact. Alternatively, theinterior of the biocatalyst immobilization chamber 43 might be coatedwith an appropriate lining material to provide the same effect.

FIG. 34 is a transverse sectional view through the rotor body 20 of FIG.31 and the rotating shaft 21 of FIG. 31 parallel to the axis ofrotation. The output liquid transport lines 53 are metal tubes whichcommunicate with the bioreactor chambers 26 and the centrally-locatedaxial liquid output channel 51 through output compression fittings (41in FIG. 33). The input liquid transport lines 54 are metal tubes whichcommunicate with the bioreactor chambers 26 and the centrally-locatedaxial liquid input channel 52 through input compression fittings (44 inFIG. 33).

FIG. 35 is a view of the rotating shaft 21 of FIG. 31 on which the rotorbody 20, the liquid output mechanical end-face seal (27 in FIG. 31), andthe liquid input mechanical end-face seal (28 in FIG. 31) are mounted.The rotating shaft 21 contains a centrally-located axial liquid outputchannel 51 and a centrally-located axial liquid input channel 52. Thecentrally-located axial liquid output channel 51 (typically ⅛″ diameter)transports the liquid output of the bioreactor chambers (26 in FIG. 31)to the liquid output mechanical end-face seal (27 in FIG. 31) by meansof three short radially-directed passages 60 while the centrally-locatedaxial liquid input channel 52 (also ⅛″ dia.) conveys liquid from theliquid input mechanical end-face seal (28 in FIG. 31) into thebioreactor chambers (26 in FIG. 31), also by means of three shortradially-directed passages 61. The centrally-located axial liquid outputchannel 51 and the centrally-located axial liquid input channel 52extend from each end of the rotating shaft 21 to the region where therotor body 20 is located. Each end of the rotating shaft 21 has athreaded recess 62 which is formed to accept threaded liquid mechanicalseals. The leftmost end of the rotating shaft 21 is also machined toprovide a keyway 63 to which a motor drive pulley (not shown) may beattached.

FIG. 36 is a view of a typical liquid output mechanical end-face sealassembly such as the liquid output mechanical end-face seal 27, shown inFIG. 31. The rotating part 72 of the liquid output mechanical end-faceseal 27 is threaded into the threaded recess (62 in FIG. 35) in theleftmost end of the rotating shaft (21 in FIG. 35). A seal between therotating and stationary portions of the liquid output mechanicalend-face seal 27 is provided by the contact of the stationary seal face70 against the rotating seal face 71. In the case of high performancemechanical end-face seals utilizable in the process of this invention,where consideration must be made for the resultant centrifugal forceswhich act on the seal components, all spring elements are located in thestationary portion of the seal assembly. While the seal assembly shownin FIG. 36 is a single seal, double and/or tandem end-face sealconfigurations may prove more advantageous in prolonged usage. When sucha seal assembly is mounted on the rotating shaft (21 in FIG. 34) of thepresent invention, aqueous liquids may be pumped out of the stationarypart 73 of the liquid output mechanical end-face seal assembly viacompression fittings and the pumped liquid will follow the pathindicated by the dotted line 74 to make communication with thecentrally-located axial liquid output channel (51 in FIG. 34) whichtransports the liquid away from the bioreactor chambers (26 in FIG. 34)mounted in the rotor body (20 in FIG. 34).

The principal disadvantage heretofore in the employment of mechanicalseals for the transfer of liquids into and out of rotating systems,where the purpose of the system is to culture biological entities suchas animal cells or micro-organisms, has been the problem of themaintenance of sterility. Low pressure mechanical seals have, in thepast, provided a route by which adventitious micro-organisms can gainentrance into bioreactor systems via the thin film of internal liquidwhich lubricates the end-face seal surfaces. In the process of thisinvention, where the internal liquid is always held at a hydraulicpressure higher than ambient, all leakage of liquid will occur to theexterior of the system; there is thus no possible route through whichadventitious contaminants can enter the system.

In order to obtain data for an analysis of the performance of a rotorbody (20 in FIG. 31) of the dimensions and configuration outlined inFIGS. 31-32 and 34-35 and containing demountable rectilinear biocatalystimmobilization chambers 43 like those depicted in FIG. 33, it wasnecessary that several scale dimensions and boundary equations be chosenarbitrarily and used to determine the operating characteristics of thesecond embodiment of the present invention. The immobilization boundaryequations chosen are those listed in Equations 1 and 2 of FIG. 15. Therotor dimensions chosen for this example and indicated by letter inFIGS. 31-33 are as follows:

a: 10.0 cm  g: 7.0 cm m: 0.5 cm b: 36.0 cm  j: 10.0 cm  n: 1.0 cm d: 4.0cm k: 1.5 cm o: 1.0 cm f: 6.0 cm L: 7.0 cm

In the first and second embodiments of this invention, described above,a portion of the geometry of the biocatalyst immobilization chamber (43in FIGS. 21 and 33) is that of a truncated cone. As is shown in FIG. 37,the dimensional problem of determining the “aspect ratio” (the ratio ofthe small radius of the truncated cone 110 to the large radius of thetruncated cone) of the biocatalyst immobilization chamber (43 in FIGS.21 and 33) due to boundary condition constraints can be reduced to anexamination of the geometrical relationships between the large and smallradii of the truncated cone 110 and the height of the truncated cone110.

FIG. 37A is a sectional view, through the plane of rotation, of theportion of the biocatalyst immobilization chamber (43 in FIGS. 21 and33) which resembles a truncated cone 110. The truncated cone 110 has aproximal face which is located a distance of R_(x) from the center ofrotation. The truncated length of the cone is R_(c). A RelativeCentrifugal Force (RCF) acts to cause translation of a particle 111 inthe biocatalyst immobilization chamber (43 in FIGS. 21 and 33) to longerradii, while liquid flow forces (FV) act to cause translation to shorterradii. Equation (1) of FIG. 37B is an expression for the magnitude ofthe Relative Centrifugal Force (RCF) at radial length (R_(x)) in termsof the Rotor Speed (RS). Equation (2) is an expression for the magnitudeof the Flow Velocity (FV) at radial length (R_(x)) in terms of theliquid Flow Rate (FR) and the large radius (q) of the truncated cone110. Equation (3) is an expression for the magnitude of the RelativeCentrifugal Force (RCF) at radial length (R_(x)+R_(c)) in terms of theRotor Speed (RS). Equation (4) is an expression for the magnitude of theFlow Velocity (FV), at radial length (R_(x)+R_(c)), in terms of theliquid Flow Rate (FR) and the given dimensions of the truncated cone 110and its sections. In order to determine the “aspect ratio” of thetruncated cone 110 which will satisfy certain boundary conditions, giventhe physical dimensions of the rotor body (20 in FIGS. 19 and 31), wehave chosen to express the radius of the small end (R1) of the truncatedcone 110 in terms of the length (L) of a non-truncated version of thetruncated cone 110. This non-truncated version of the truncated cone 110is shown in dotted lines in FIG. 37B.

The desired boundary conditions are: (1) that the product of theintrinsic Sedimentation Rate (SR) of the immobilized particle due togravity and the applied centrifugal field (RCF) be exactly equal to themagnitude of the liquid flow forces (FV) at the most distal portion ofthe biocatalyst immobilization chamber (43 in FIGS. 21 and 33); and (2)that this product be twice the magnitude of the liquid flow forces (FV)at the most proximal portion of the biocatalyst immobilization chamber(43 in FIGS. 21 and 33). Thus:

at centrifugal radius=R _(x) +R _(c)(SR)×(RCF)=FV

at centrifugal radius=R _(x)(SR)×(RCF)=2×FV

Substituting into these equations the dimensional specifications for RCFand FV obtained from Eqns. (1-4) of FIG. 37, we now have twosimultaneous equations which relate the liquid Flow Rate (FR), the RotorSpeed (RS), and the dimensions of the biocatalyst immobilization chamber(43 in FIGS. 21 and 33):${(1)\quad ({SR})\quad {C_{1}\left( {R_{X} + R_{C}} \right)}} = {C_{2}\left( \frac{L}{L - R_{C}} \right)}^{2}$

 (SR)C ₁(R _(x))=2×C ₂  (2)

In order to arrive at a solution to these equations, we will make thefollowing substitutions which are based on the physical dimensionallimits of the example rotor system:

R _(x)=90 mm

R _(c)=30 mm

q=30 mm

The simultaneous equations now become:${(1)\quad ({SR})\quad {C_{1}(120)}} = {C_{2}\left( \frac{L}{L - 30} \right)}^{2}$

 (SR)C ₁(R _(x))=2×C ₂  (2)

Substituting Eqn. (2) into Eqn. (1) yields:$\left( \frac{L}{L - 30} \right)^{2} = {240/90}$

Solution of this quadratic expression yields L=77.4 mm and:$\left( \frac{L}{L - 30} \right)^{2} = 2.67$

Since it was earlier determined (see FIG. 37) that:$R_{1} = \frac{q\left( {L - R_{c}} \right)}{L}$

Thus, the smaller radius of the truncated cone which satisfies theboundary conditions is:

R ₁=18.4 mm

Now, the two simultaneous equations become:

(SR)C ₁(120)=C ₂(2.67)  (1)

(SR)C ₁(90)=C ₂(2)  (2)

and, by subtracting (2) from (1) and collecting terms, we arrive at:

(SR)(30)C ₁=(0.67)C ₂  (3)

Substitution into (3) of the values of C₁ and C₂ yields:${(3)\quad ({SR})\quad (30)\quad (1.12)\quad \left( \frac{RPM}{1000} \right)^{2}} = {(0.67)\quad \left( \frac{FR}{\pi \cdot q^{2}} \right)}$

Now we have an expression which satisfies the desired boundaryconditions and physical dimensional constraints in terms of thecontrollable variables, RS and FR:

{square root over (SR)}(RPM)=(2.65){square root over (FR)}

Thus, once the physical dimensions of the rotor system as well as thoseof the biocatalyst immobilization chamber have been determined, therange of Rotor Speeds (RS) and the system liquid Flow Rates (FR) whichwill constrain the particles to immobility in the bioreactor will followa simple relationship which is dependent only on the intrinsicSedimentation Rate (SR) of the object particle due to gravity. Notethat, under the above conditions, the maximal volume of immobilizationis ca. 56 mL per bioreactor chamber.

This method and apparatus for containing a biocatalyst comprises thestep of containing the biocatalyst in a bioreactor chamber placed in acentrifugal force field where the centrifugal force field is oriented ina plane parallel to the plane in which the force of gravity acts. Thecentrifugal force field is diametrically opposed by a continuouslyflowing liquid at hydraulic pressures greater than the ambientbarometric pressure.

FIG. 45 depicts the components of a third embodiment of this invention.This embodiment is a design which may be employed for applications wherethe immobilized biocatalyst is in a complex consisting of a dense inertsupport particle to which the actual biocatalyst is attached. In such anapplication, the buoyant force acting on the biocatalyst/support complexas a result of nutrient liquid flow can be negated, and thusimmobilizing the biocatalyst/support complex, by the vertical alignmentof the biocatalyst immobilization chamber so that the earth'sgravitational field acts on the biocatalyst/support complex to providethe required counter-acting force. Further, the range of flow rateswhich can be accommodated in this system is in no way limited since thebuoyant force which must be countered is the nutrient liquid flowvelocity. The magnitude of the flow velocity can be varied through adesired range by varying the cross-sectional diameters and the aspectratio of those diameters as necessary. The relative centrifugal field inthis case is close to 1×g (that provided by the earth's gravitationalfield). Thus, the required applied centrifugal field, in this case, iszero.

In the embodiment shown in FIG. 45, nutrient liquids, which have beenpressurized and have dissolved in this liquid the appropriate quantitiesof a nutrient which is gaseous at ambient pressure, are pumped into astationary biocatalyst immobilization chamber fed by the main feed pump,Pump 3. The continuation of the liquid flow as it exits the biocatalystimmobilization chamber is fed through control and monitoring sensors andthrough a system pressure regulator which maintains the elevatedhydraulic pressure of the system. The ratio of R₁ to R₂ is dependent onthe desired flow velocity boundary conditions and can vary downward from1.0 to any desired fraction thereof. R₁ is not limited in dimension: itsmagnitude is determined by the size of the liquid flow rate which isdesired. L, the height of the immobilization chamber, is not limited indimension: its magnitude is determined by the desired retention time ofa nutrient liquid bolus as it passes through the biocatalystimmobilization chamber.

In order to obtain data for an analysis of the performance of abiocatalyst immobilization chamber of the embodiment in FIG. 45,(dimensions denoted by letters), it was necessary that several scaledimensions and boundary equations be chosen arbitrarily and used todetermine the operating characteristics of an embodiment of the presentinvention. The immobilization boundary equations (both the top andbottom boundary equation) chosen is that listed in Equation 1 of FIG.15. The biocatalyst immobilization chamber dimensions chosen for thisexample and indicated by letter in FIG. 45 are as follows:

R ₁:5.0 cm

R ₂:5.1 cm

L:61.0 cm

A biocatalyst immobilization chamber of the above dimensions was loadedwith 100 mL of 30-50 mesh peanut shell charcoal (density: ca. 3.5gm/mL). The system configuration of FIG. 45 was established and, at aliquid flow rate of 120 mL/min, an equilibrium between the flowvelocity-derived buoyant forces and the intrinsic sedimentation rate ofthe individual charcoal particles at 1×g relative gravitational fieldresulted in a stable, immobilized, three-dimensional array. Note thatsmall flow rate variations near the nominal value chosen resulted insmall increases or decreases in the immobilized array density andvolume, while large changes in flow rate require that R₁, R₂, and L bechanged, thus requiring separate biocatalyst immobilization chambersizes to accommodate different flow rate regimes. While the charcoalparticles were found suitable for the attachment of a number ofbacterial genera, the type of inert particle employed for a specificbiocatalyst immobilization purpose are limited only in the compatabilitywith the biocatalyst and the liquid environment of the system.

There are many alternative shapes for the biocatalyst immobilizationchambers which are contemplated in this invention. One such alternativeembodiment is a biocatalyst immobilization chamber having its innerspace in the shape of a right circular cone with a major axis which isaligned parallel to the applied centrifugal force field and which has alarge diameter which is nearer to the axis of rotation than is its apex.

Another alternative embodiment is a biocatalyst immobilization chamberhaving its inner space in the shape of a right circular cone which has amajor axis which forms an angle of between 0 and 90 degrees with theapplied centrifugal force field. Also included in the present inventionis a biocatalyst immobilization chamber having its inner space in theshape of a truncated right circular cone which has a major axis which isaligned parallel to the applied centrifugal force field and which has alarge diameter which is nearer to the axis of rotation than is its minordiameter.

Additionally, the present invention includes a biocatalystimmobilization chamber having its inner space in the shape of atruncated right circular cone which has a major axis which forms anangle of between 0 and 90 degrees with the applied centrifugal forcefield.

The present invention includes a biocatalyst immobilization chamberhaving its inner space in the shape of a sphere where the appliedcentrifugal force field is perpendicular to a circular cross-section ofthe spherical biocatalyst immobilization chamber.

The present invention also includes a biocatalyst immobilization chamberhaving its inner space in the shape of a sphere where the appliedcentrifugal force field forms an angle of between 0 and 90 degrees withthe circular cross-section.

Additionally, the present invention includes a biocatalystimmobilization chamber having its inner space is in the shape of atruncated sphere where the applied centrifugal force field isperpendicular to a circular cross-section of the sphere.

The present invention also includes a biocatalyst immobilization chamberhaving its inner space in the shape of a truncated sphere where theapplied centrifugal force field forms an angle of between 0 and 90degrees with the circular cross-section.

The present invention includes a biocatalyst immobilization chamberhaving its inner space in a shape which possesses a varying circularcross-section where the applied centrifugal force field is perpendicularto the circular cross-sections.

The present invention also includes a biocatalyst immobilization chamberhaving its inner space in a shape which possesses a varying circularcross-section where the applied centrifugal force field forms an angleof between 0 and 90 degrees with the circular cross-sections.

Additionally, the present invention includes a biocatalystimmobilization chamber having its inner space in a shape which possessesa varying elliptical cross-section where the applied centrifugal forcefield is perpendicular to the elliptical cross-sections.

The present invention also includes a biocatalyst immobilization chamberhaving its inner space in a shape which possesses a varying ellipticalcross-section where the applied centrifugal force field forms an angleof between 0 and 90 degrees with the elliptical cross-sections.

Also included in present invention is a biocatalyst immobilizationchamber having its inner space in a shape which is a combination ofcircular and elliptical cross-sections along an axis perpendicular tothe applied centrifugal force field.

The present invention also includes a biocatalyst immobilization chamberhaving its inner space in a shape which is a combination of circular andelliptical cross-sections along an axis which forms an angle of between0 and 90 degrees to the circular and/or elliptical cross-sections.

The process of this invention is directed toward the immobilization ofbiocatalysts such as micro-organisms and eukaryotic cells, theirsubcellular organelles, and natural or artificial aggregates of suchbiocatalysts. Thus, the process system must be capable of immobilizingfairly light particles. It is known that the sedimentation rates of suchparticles due to gravity range from ca. 0.01 mm/min for small bacteriato 0.1 mm/min for small animal cells to more than 10.0 mm/min forthick-walled micro-organisms (such as yeasts) and biocatalyticaggregates such as bead-immobilized cells. We have analyzed theperformance characteristics of the centrifugal bioreactor system of thisinvention using the dimensional configurations outlined above andpresent these results below.

FIG. 38 displays profiles of the values of rotor speed and liquid flowrate which satisfy the boundary conditions outlined earlier for therotor and bioreactor dimensions outlined in FIGS. 19-29 (for the firstembodiment of the present invention), and in FIGS. 31-35 (for the secondembodiment of the present invention) for typical biologicallysignificant particles of the lowest two Sedimentation Rate (SR) ranges.The upper line displays the continuum of liquid flow rates and rotorspeeds which result in the immobilization of particles of an intrinsicSedimentation Rate (SR) of 0.001 mm/min, a value smaller by a factor often than any we have measured for any tested micro-organism. Note that,even at a flow rate of 10 mL/min, the rotor speed required to maintainimmobilization is a physically reasonable value and that the maximumcentrifugal force (RCF) required is ca. 9400×g, a value well within thephysical limits of average quality centrifugal systems. The lower linedisplays the corresponding profile for particles of a Sedimentation Rate(SR) of 0.01 mm/min, a value near that exhibited by typicalrepresentative bacteria. Again, this line represents a continuum ofvalues which satisfy the immobilization conditions. Thus for example, ifa flow rate of 2.0 mL/min is required to adequately nutrition aparticular sized three-dimensional array of “bacteria A,” a rotor speednear 1200 rpm will suffice, while a required flow rate of 8.0 mL/minnecessitates a rotor speed near 2500 rpm. Note that the heavierparticles of SR=0.01 mm/min require only a modest maximal centrifugalforce of ca. 1000×g at a flow rate of 10.0 mL/min.

FIG. 39 displays profiles of the values of rotor speed and liquid flowrate which satisfy the boundary conditions outlined earlier for therotor and bioreactor dimensions outlined in FIGS. 19-29 and 31-35 in thecases of typical biologically significant particles of the higher threeSedimentation Rate (SR) ranges. The upper line displays the continuum ofliquid flow rates and rotor speeds which result in the immobilization ofparticles comparable to larger micro-organisms or small animal cells(for example, mammalian erythrocytes) of an intrinsic Sedimentation Rate(SR) of 0.1 mm/min. The middle line displays the corresponding valuesfor the immobilization of more typical animal cells (ca. 30 μm diameter;SR=1.0 mm/min), while the bottom line displays the continuum of valueswhich provide for the immobilization of large dense cells, such aseukaryotic yeasts (SR=10 mm/min). As was the trend shown in FIG. 38, itis obvious from the data of FIG. 39 that the maximum rotor speeds andmaximal centrifugal forces required in this flow rate range decrease asthe intrinsic particle Sedimentation Rate (SR) due to gravity increases.Thus, for a flow rate of 10.0 mL/min, a three-dimensional array ofaverage-sized animal cells requires only a relative centrifugal force ofca. 10×g to provide immobilization.

FIG. 40 displays profiles of the values of rotor speed and liquid flowrate which satisfy the boundary conditions outlined earlier for therotor and bioreactor dimensions outlined in FIGS. 19-29 and FIGS. 31-35in the case of liquid flow rates of as much as 100 mL/min for thehighest three intrinsic Sedimentation Rate (SR) ranges examined. Thus,even if the liquid flow rates required to nutrition such immobilized“beds” of particles (example bed volume=56 mL) is increased ten-fold,the maximal centrifugal forces and rotor speeds required are technicallyunremarkable. Note that, in the case of “animal cells” (SR=1.0 mm/min) aflow rate of 100 mL/min represents a flow of 6.0 L/hr, a flow obviouslylarger than that required to adequately nutrition such athree-dimensional array of cells under any imaginable conditions.

While it is generally obvious that the effect of immobilizing apopulation of, for example, bacteria in a flowing liquid will not leadto cellular damage as a result of the flow of liquid past the surface ofsuch cells (since many micro-organisms possess extracellular “sheaths”which protect their plasma membranes from liquid shear forces), it isless obvious whether delicate animal cells (which do not possess suchextracellular protection) would remain viable under these conditions.However, as was shown in FIG. 39, the maximum Relative Centrifugal Force(RCF) required to maintain an average-sized animal cell immobile in aliquid flow of 10 mL/min is ca. 10×g. Even if this flow is raised to alevel decidedly well above any anticipated nutritional need (100mL/min), the maximum RCF required is only ca. 100×g (FIG. 40). It shouldbe remembered that the immobilization of such a cell in a flowing liquidis the mathematical equivalent of moving the cell through a stationaryliquid. Thus, since the conventional laboratory sedimentation of animalcells through liquid media at RCF's of more than 100×g is anunremarkable phenomenon, it is unlikely that the shear forces acting onsuch cells in the process of this invention will cause any damage totheir plasma membranes. This assertion is supported by the operatingcharacteristics of a related device, the Beckman JE-5.0 CentrifugalElutriation System, from which viable animal cells have beensuccessfully recovered after exposure to flow rates and RCF's greatly inexcess of those proposed herein for the process of this invention.

With the present invention, it is possible to immobilizethree-dimensional arrays of biologically-significant particles and toadequately nutrition the immobilized particles with a completely liquidflow. In particular, for the small-scale prototypic centrifugal processoutlined above, the required centrifugal forces and liquid flow ratesare not unusual and present no novel problems such as, for example,requiring unreasonably high rotational speeds or flow rates. Further, ithas been demonstrated that there is a wide range of paired flow rate andangular velocity values which maintain the immobilization ofthree-dimensional arrays of such particles.

The fact that there is a wide range of flow rates and correspondingrotational speeds which can be used to immobilize such arrays ofparticles has, however, a wider significance. Using conventional culturemethodology, the major problem encountered in large-scale culture is theinability to adequately nutrition dense masses of metabolically-activebiological units. In the case of conventional mammalian cell culture forexample, an average cell density of more than 1×10⁶ cells/mL is rarelyachieved for prolonged time periods for this reason. Similarly,bacterial cell densities between 1×10⁷ and 1×10⁹ cells/mL are rarelyexceeded in mass culture by conventional methods for this same reason.Using the methodology of the process of this invention, as cell densityand effective “bed” volume increases (either from cellular proliferationor bioreactor loading), the increased nutritional requirements of largeror more dense cultures can be met by increasing input liquid flow whilesimultaneously increasing the size of the applied centrifugal field.Using the process of this invention, it is possible to easily maintainmammalian cell cultures at concentrations two powers of ten greater thanconventional, with bacterial cell densities approaching between 1×10¹⁰and 1×10¹¹ cells/mL equally realizable.

Similarly, for dense cultures of aerobic organisms, the conventionalproblem of adequate delivery of optimal dissolved oxygen to the cultureis easily solved using the process of this invention. Since it ispossible to dissolve molecular oxygen in typical culture media atconcentrations of more than 100 mM (using a hydraulic pressure of ca.1500 psig) the problem of the delivery of optimal dissolved oxygen, forany imaginable dense culture, is solved simply by adjusting the systemhydraulic pressure to a value which will maintain the solubility of thedesired concentration of oxygen. The ability to maintain dissolvedoxygen concentration at optimal levels results in greatly increasedproduction efficiency. As has been noted by many researchers, theinability to achieve cellular production efficiencies near thoseobserved in vivo is a major disadvantage of conventional animal culturetechniques (The Scientist, 8, #22, pg.16, Nov. 14, 1994).

The ability to achieve near-normal aerobic efficiency in dense culturehas another, less obvious, advantage; the generation of heat. Instead ofrequiring expensive energy input to bring the liquid cellularenvironment to an optimal temperature, it is likely that the pumpedliquid of the process of this invention will have to be delivered to thecellular environment at reduced temperatures in order to carry awayexcess metabolic heat.

Another important advantage of the process of this invention is therelative invariance of the chemical composition of the liquidenvironment in which the three-dimensional arrays of biocatalysts areimmobilized. Since the arrays are continually presented with fresh,optimal liquid nutrient input and since these arrays are continuallydrained by the continuance of the process flow, the chemical compositionof the cellular environment will be completely invariant in time. Therewill be shallow chemical gradients of nutrients, product(s), andmetabolites across the radial length of these arrays, but since theradial length is the shortest dimension of the array, these gradientswill be minimal and can be easily compensated for by tailoring the mediacomposition. Thus, for example, a pH change across the array depth canbe compensated for with minimal buffering while input nutrient gradientsacross the array depth can be similarly compensated for.

The most important advantage of the process of the present invention,however, is the fact that metabolic waste products will be continuallyremoved from the cellular environments by the liquid process flow. Sinceit has been suggested that the inability to remove metabolic wastes andthe inability to continually remove desired products from the cellularenvironment is a major factor in lowered per-cell productivity, it islikely that the utilization of the process of this invention willmarkedly increase general cellular productivity.

The chemical composition of optimal input liquid nutrient media toimmobilized populations of biocatalysts in the process of this inventionwill be quite different from that of conventional nutrient media. Inparticular, the optimal media composition in this process will be thatwhich can be completely consumed in one pass through the bioreactorchamber. Typical nutrient media contain a mix of as many as thirty ormore nutrient chemicals, all of which are present in amounts whichgreatly exceed the nutritional needs of the biocatalysts. This isbecause the nutrient media must sustain their metabolic processes for aslong as 100 hours in some cases. Similarly, conventional media containconcentrations of pH buffer compounds and indicators and hormonalstimuli (fetal sera and/or cytokines, etc.) in amounts which greatlyexceed the immediate needs of the biocatalysts. In the process of thisinvention, the input liquid medium can be tailored to contain thoseconcentrations of nutrients and stimulants which are directly requiredby the immediate metabolism of the immobilized biocatalysts. Ideally,the outflowing liquid which exits the bioreactor would be completelydevoid of nutrients and contain only salts, metabolic wastes, andproduct molecules. The present invention makes it possible to tailor theinput media in order to maintain an immobilized cellular population in anutritional state which either promotes or inhibits cellularproliferation. It is highly unlikely that a nutritional mix which isoptimal for cellular division is optimal for the production ofbiochemicals by cells at rest in the cell cycle.

The liquid medium used in the present invention may be any formulationknown to those skilled in the art or may include specific individualcomponents which are necessary for the biocatalyst of interest. Thekinds of media may include, but are not limited to, a nutrient medium, abalanced salt solution, or a medium containing one or more organicsolvents. The medium may contain dissolved gases for growth of thebiocatalyst under anaerobic or aerobic conditions. The medium may beformulated so that the biocatalyst product or mobile biocatalysts foundin the medium are more easily isolated.

Another less obvious implication of the utility of this processmethodology is the effect of scaling. In the first and secondembodiments of the present invention, the total volume capacity of thefour-bioreactor rotor is ca. 224 mL and 170 mL, respectively. Note,however, that as the radius of the rotor is increased, the volumecapacity of the system goes up as the cube of the radius. This is shownin the graph of FIG. 41, in which the leftmost point corresponds to thefirst embodiment of this invention, and in FIG. 42, in which theleftmost point corresponds to the second embodiment of this invention. Arotor with a radius of 1.5 meters would have a volume capacity of ca.120 liters. Further, since the average density of culture is roughly 100times that of conventional culture methods, the equivalent culturevolume is proportionally larger. Thus, a centrifugal fermentation unitwith a rotor radius of 1.5 m is roughly equivalent to a 12,000 literfermentation using current technology.

Finally, it should be noted that there is an additional advantage inscale in the use of the process of this invention. As a consequence ofthe fact that relative centrifugal force is directly proportional to therotor radius but is also directly proportional to the square of theangular velocity, the rotational speeds required to maintain a desiredrelative centrifugal force decrease as the rotor radius is increased.This is shown graphically in FIG. 43. While the rotational speedrequired to maintain a RCF=100×g is ca. 810 rpm for a rotor with aradius of 18 cm, this required rotational speed drops to less than 300rpm when the rotational radius is increased to 1.5 m. This is more thana 50% lowering in the speed of rotation.

While it is obvious that scale-up of this process will have value inindustrial production facilities, it should be noted that a miniatureembodiment of the Centrifugal Fermentation Process could be valuable inthe analytical study of the “metabolic physiology” of small homogeneouspopulations of a particular cell type. To our knowledge, the exactnutritional requirements for maximal proliferation of, for example, abacterial population are unknown—and could be rapidly and easilydetermined by perturbation of the composition of the nutritional liquidinput to an immobilized test population while measuring some outputparameter indicative of growth. Similarly, while it is desirable to knowexactly what nutritional mix is optimal for cellular production of abiological product (a nutritional mix which is highly unlikely to beidentical to that which maximizes proliferation), such parameters are,again, unknown. We believe that small-scale versions of the process ofthis invention could be advantageously utilized in advancing “analyticalmicrobiology” or “analytical cell biology” in a fashion heretoforeimpossible to perform.

The present invention may also be used for the continuous production ofbiological products which are secreted or otherwise released into theout-flowing liquid stream. Thus, for example, one might utilize thisprocess for the continual harvest of product(s) which are released froman immobilized micro-organism population whose growth rate (and deathrate) have been nutritionally manipulated to maintain a steady stateimmobilized “bed volume”. Such a process could run, theoretically,forever. Similarly, the immobilization of secretory animal cellpopulations would result in continual outflow of liquid enriched in thedesired product(s).

The present invention is also extremely useful in the creation ofnon-secreted products (such as the cytosolic accumulation of protein ingenetically-engineered E. coli). If an immobilized cell population ismaintained in the bioreactor system outlined above, but under conditionsof excess nutritional input, then the population will quickly grow to anenlarged bed size which will continually “leak” excess cells into theout-flowing liquid stream. Thus, the process of this invention can beoperated as a “production cow.” That is, the present invention can beused as a continual incubator for the production and outflow of maturecells which are rich in the desired product. Downstream isolation anddisruption of the out-flowing cell stream to capture the product ofinterest would then follow conventional product purification methods.

The process of this invention offers the possibility of continual,serial interconversion of bio-organic substrates through severalintermediate steps by two or more separate animal cell populations ormicro-organism populations. As a consequence of the ability of theprocess of this invention to completely immobilize biocatalystpopulations while continually flowing a liquid stream into and out ofthe immobilized population, it now becomes possible to serially connectseparate, disparate immobilized populations into one flowing processstream with the assurance that there will be no cross-contamination ofone population with the other. To accomplish this, several of thedevices described herein are connected in series so that materials flowfrom one device into another device and then into the following deviceand so on. As is shown in FIG. 44, a process flow schematic in which abiochemical substrate, which is provided as a dissolved nutrient in theprimary media reservoir, is converted into intermediate “product A” byits passage through the biocatalyst population immobilized in Centrifugeand Rotor #1 and is then further converted into “product B ” by passagethrough a biocatalyst population immobilized in Centrifuge and Rotor #2.Furthermore, it is possible to change the composition of the liquidnutritional feedstock between the two immobilized populations sinceneither centrifuge/rotor combination is constrained to operate at thesame flow rate and angular velocity as the other. Thus, as is shown inFIG. 44, the liquid flow into Centrifuge and Rotor #2 may be modified bymeans of an additional pump supplying necessary nutrients from MediaReservoir #2; the total flow per unit time through Centrifuge and Rotor#2 is simply higher than that through Centrifuge and Rotor #1.

A commercially-valuable example of the utility of a serial conversionprocess of this type is the biological production of acetic acid.Anaerobic bio-conversion of glucose into ethanol by an immobilizedpopulation of a yeast such as Saccharomyces cerevisiae in Centrifuge andRotor #1 could be followed by aerobic conversion of ethanol to aceticacid by an immobilized population of the bacterium Acetobacter acetiilocated in Centrifuge and Rotor #2. This would require that dissolvedoxygen and supplemental nutrients be provided via Media Reservoir #2(using, for example, the oxygenation scheme depicted in FIG. 17).

Similarly, if a process flow scheme demanded that total flow volume perunit time through specific centrifugal bioreactor units be reduced, thena series of identical centrifugal bioreactor units could be connected inparallel to the process stream flow, with the resultant individual flowvolume per unit time thereby reduced to the fractional flow through eachunit. In this case, the devices of the present invention would beconnected in a parallel arrangement.

The microbial organisms which may be used in the present inventioninclude, but are not limited to, dried cells or wet cells harvested frombroth by centrifugation or filtration. These microbial cells areclassified into the following groups: bacteria, actinomycetes, fungi,yeast, and algae. Bacteria of the first group, belonging to ClassShizomycetes taxonomically, are Genera Pseudomonas, Acetobacter,Gluconobacter, Bacillus, Corynebacterium, Lactobacillus, Leuconostoc,Streptococcus, Clostridium, Brevibacterium, Arthrobacter, or Erwinia,etc. (see R. E. Buchran and N. E. Gibbons, Bergey's Manual ofDeterminative Bacteriology, 8th ed., (1974), Williams and Wilkins Co.).Actinomycetes of the second group, belonging to Class Shizomycetestaxonomically, are Genera Streptomyces, Nocardia, or Mycobacterium, etc.(see R. E. Buchran and N. E. Gibbons, Bergey's Manual of DeterminativeBacteriology, 8th ed., (1974), Williams and Wilkins Co.). Fungi of thethird group, belonging to Classes Phycomycetes, Ascomycetes, Fungiimperfecti, and Bacidiomycetes taxonomically, are Genera Mucor,Rhizopus, Aspergillus, Penicillium, Monascus, or Neurosporium, etc. (seeJ. A. von Ark, “The Genera of Fungi Sporulating in Pure Culture”, inIllustrated Genera of Imperfect Fungi, 3rd ed., V. von J. Cramer, H. L.Barnett, and B. B. Hunter, eds. (1970), Burgess Co.). Yeasts of thefourth group, belonging to Class Ascomycetes taxonomically, are GeneraSaccharomyces, Zygosaccharomyces, Pichia, Hansenula, Candida,Torulopsis, Rhodotorula, Kloechera, etc. (see J. Lodder, The Yeasts: ATaxonomic Study, 2nd ed., (1970), North-Holland). Algae of the fifthgroup include green algae belonging to Genera Chlorella and Scedesmusand blue-green algae belonging to Genus Spirulina (see H. Tamiya,Studies on Microalgae and Photosynthetic Bacteria, (1963) Univ. TokyoPress). It is to be understood that the foregoing listing ofmicro-organisms is meant to be merely representative of the types ofmicro-organisms that can be used in the fermentation process accordingto the present invention.

The culture process of the present invention is also adaptable to plantor animal cells which can be grown either in monolayers or in suspensionculture. The cell types include, but are not limited to, primary andsecondary cell cultures, and diploid or heteroploid cell lines. Othercells which can be employed for the purpose of virus propagation andharvest are also suitable. Cells such as hybridomas, neoplastic cells,and transformed and untransformed cell lines are also suitable. Primarycultures taken from embryonic, adult, or tumorous tissues, as well ascells of established cell lines can also be employed. Examples oftypical such cells include, but are not limited to, primary rhesusmonkey kidney cells (MK-2), baby hamster kidney cells (BHK21), pigkidney cells (IBRS2), embryonic rabbit kidney cells, mouse embryofibroblasts, mouse renal adenocarcinoma cells (RAG), mouse medullarytumor cells (MPC-11), mouse-mouse hybridoma cells (I-15 2F9), humandiploid fibroblast cells (FS-4 or AG 1523), human liver adenocarcinomacells (SK-HEP-1), normal human lymphocytic cells, normal human lungembryo fibroblasts (HEL 299), WI 38 or WI 26 human embryonic lungfibroblasts, HEP No. 2 human epidermoid carcinoma cells, HeLa cervicalcarcinoma cells, primary and secondary chick fibroblasts, and variouscell types transformed with, for example, SV-40 or polyoma viruses (WI38 VA 13, WI 26 VA 4, TCMK-1, SV3T3, etc.). Other suitable establishedcell lines employable in the method of the present invention will beapparent to the person of ordinary skill in the art.

The products that can be obtained by practicing the present inventionare any metabolic product that is the result of the culturing of a cell,either eukaryotic or prokaryotic; a cell subcellular organelle orcomponent, such as mitochondria, nuclei, lysozomes, endoplasmicreticulum, golgi bodies, peroxisomes, or plasma membranes orcombinations thereof; or an enzyme complex, either a natural complex ora synthetic complex, i.e., a plurality of enzymes complexed together toobtain a desired product.

One of the advantages of the present invention is the ability to producea desired chemical from a cell without having to go through thelaborious process of isolating the gene for the chemical and theninserting the gene into a suitable host cell, so that the cell (and thusthe chemical) can be produced in commercial quantities. The presentinvention may be used to directly culture, in high-density, a mammaliancell that is known to produce a desired chemical. By doing this, thepresent invention may be used to produce large quantities of the desiredchemical.

Products that can be produced according the present invention include,but are not limited to, immunomodulators, such as interferons,interleukins, growth factors, such as erythropoietin; monoclonalantibodies; antibiotics from micro-organisms; coagulation proteins, suchas Factor VIII; fibrinolytic proteins, such as tissue plasminogenactivator and plasminogen activator inhibitors; angiogenic proteins; andhormones, such as growth hormone, prolactin, glucagon, and insulin.

The term “culture medium” includes any medium for the optimal growth ofmicrobial, plant, or animal cells or any medium for enzyme reactionsincluding, but not limited to, enzyme substrates, cofactors, buffers,and the like necessary for the optimal reaction of the enzyme or enzymesystem of choice. Suitable culture media for cell growth will containassimilable sources of nitrogen, carbon, and inorganic salts, and mayalso contain buffers, indicators, or antibiotics.

Any culture medium known to be optimal for the culture ofmicroorganisms, cells, or biocatalysts may be used in the presentinvention. While such media are generally aqueous in nature for theculture of living organisms, organic solvents or miscible combinationsof water and organic solvents, such as dimethylformamide, methanol,diethyl ether and the like, may be employed in those processes for whichthey are proved efficacious, such as those bioconversions in whichimmobilized biocatalysts are employed. Passage of the liquid mediathrough the process system may be either one-pass or the liquid flow maybe recycled through the system for higher efficiency of conversion ofsubstrate to product. Desired nutrients and stimulatory chemicals may beintroduced into the process flow, either via the low pressure nutrientsupply or via injection into the process flow upstream of the cellchamber.

It will be appreciated that the present invention is adaptable to any ofthe well-known tissue culture media including, but not limited to, BasalMedium Eagle's (BME), Eagle's Minimum Essential Medium (MEM), Dulbecco'sModified Eagle's Medium (DMEM), Ventrex Medium, Roswell Park Medium(RPMI 1640), Medium 199, Ham's F-10, Iscove's Modified Dulbecco Medium,phosphate buffered salts medium (PBS), and Earle's or Hank's BalancedSalt Solution (BSS) fortified with various nutrients. These arecommercially-available tissue culture media and are described in detailby H. J. Morton (1970) In Vitro 6, 89-108. These conventional culturemedia contain known essential amino acids, mineral salts, vitamins, andcarbohydrates. They are also frequently fortified with hormones such asinsulin, and mammalian sera, including, but not limited to, bovine calfserum as well as bacteriostatic and fungistatic antibiotics.

Although cell growth or cell respiration within the biocatalystimmobilization chamber cannot be directly visualized, such metabolismmay be readily monitored by the chemical sensing of substrate depletion,dissolved oxygen content, carbon dioxide production, or the like. Thus,for example in the case of a fermentation of a species of Saccharomycescerevisiae, inoculation of the biocatalyst immobilization chamber with asmall starter population of cells can be followed by an aerobicfermentation regime in which glucose depletion, dissolved oxygendepletion, and carbon dioxide production across the biocatalystimmobilization chamber are measured either chemically or via appropriatesensing electrodes. Thus, cell replication can be allowed to proceeduntil an optimal cell bed size is reached. Withdrawal of dissolvedoxygen input at this time causes the immobilized yeast cells to shiftinto anaerobic fermentation of glucose with a resultant production ofethanol, a process which can likewise be monitored chemically.

Similarly, without any process modification, the process of the presentinvention can be utilized as a bioreactor for immobilized chemicalcatalysts, enzymes or enzyme systems. In such a process, a catalyst, anenzyme or an enzyme system is chemically immobilized on a solid supportincluding, but not limited to, diatomaceous earth, silica, alumina,ceramic beads, charcoal, or polymeric or glass beads which are thenintroduced into the biocatalyst immobilization chamber. The reactionmedium, either aqueous, organic, or mixed aqueous and organic solvents,flows through the process system and through the three-dimensional arrayof solid supports within the bioreactor. The catalyst, enzyme, or enzymesystem converts a reactant in the process flow medium into the desiredproduct or products. Similarly, in other applications, either cells orcell components including, but not limited to, vectors, plasmids, ornucleic acid sequences (RNA or DNA) or the like may be immobilized on asolid support matrix and confined for similar utilization in convertingan introduced reactant into a desired product.

Commercial application of the present invention can be in the productionof medically-relevant, cellularly-derived molecules including, but notlimited to, anti-tumor factors, hormones, therapeutic enzymes, viralantigens, antibiotics and interferons. Examples of possible productmolecules which might be advantageously prepared using the method of thepresent invention include, but are not limited to, bovine growthhormone, prolactin, and human growth hormone from pituitary cells,plasminogen activator from kidney cells, hepatitis-A antigen fromcultured liver cells, viral vaccines and antibodies from hybridomacells, insulin, angiogenisis factors, fibronectin, HCG, lymphokines,IgG, etc. Other products will be apparent to a person of ordinary skillin the art.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE I Removal of Heavy Metals from Aqueous Solution

Microbial populations have been shown to be capable of either adsorbing,absorbing, or metabolizing a wide range of inorganic cationic complexespresented to the microbial population in dilute aqueous solution.Further, it has been amply demonstrated that virtually all terrestrial,as well as many marine, microorganisms exist in nature by attachment toa solid support through the agency of either homogeneous orheterogeneous biofilms. We demonstrate herein a novel bioremediationprocess which exploits these microbial characteristics to remove heavymetals which are presented at low concentration in very large volumeaqueous solution.

While it has thought that microbial bioremediation of aqueous heavymetal contaminants would be much cheaper and simpler than currentremediation techniques, the central drawback to their employment hasbeen the impossibility of economically processing dilute contaminants.The high flow rates which are typically required for diluteconcentrations of contaminants will “wash out” the desired microbialpopulation well before they can perform the desired bioremediation.

A microbial population is immobilized in biocatalyst immobilizationchamber or chambers. These chambers are placed into an embodiment of theapparatus shown herein, preferably in the first or second embodiment Theflow rate and rotor RPM are chosen to allow the immobilization of athree dimensional array of the chosen microorganism. Since it isessential that the pumped system has only two phases (liquid and solid),the pumped system is maintained at hydraulic pressures above ambient bymeans of a system pressure regulator downstream of the BiocatalystImmobilization Chamber(s). Nutrient minerals, organics, and dissolvedgas(es) are supplied to the Biocatalyst Immobilization Chamber(s) by themain system pump, Pump 3.

Populations of the bacterium, Pseudomonas putida, were immobilized andcultured in CBR units (RPM=850, FR=1.0 mL/min, T=37/30° C., pH=6.5,media: LB). Though not wishing to be bound by any particular theory, itwas theorized that bacteria of this genus were capable of adsorbingsignificant quantities of heavy metal ions. These experiments examinedthe ability of CBR-immobilized populations of several species to removeuranyl ion (UO₂ ²⁺) from an aqueous solution. Further, since uranylions, as groundwater contaminants, are typically found in relativelyacidic solutions (pH=4-5), all immobilized cell culture/cell adsorptionexperiments were performed in acidic solution. We found that all testedspecies can grow, albeit slowly, under acidic conditions and also adsorband internalize significant quantities of this environmental pollutant.While populations of P. aeruginosa were somewhat superior in uranyl ionuptake, we have concentrated on studying the uptake characteristics ofP. putida populations since there is a small health risk associated withP. aeruginosa handling. The results of an example experiment are shownin FIG. 46. After ca. 1×10¹⁰ P. putida cells were injected into andimmobilized in the CBR, a flow of 5 ppm uranyl nitrate (pH=4.7) wasstarted. The CBR output was monitored by ICP-AES for uranyl ionthroughput. As is shown on the figure, ca. 16 L of liquid was collectedbefore the output uranyl ion exceeded 5% of its input value, whereas theoutput uranyl ion exceeds 80% of the input value after the passage ofonly the passage of 4L in the absence of the cell population. The formerrepresents the adsorption and/or internalization of ca. 31 mg UO₂ ²⁺ byca. 826 mg of dry biomass weight.

Processing of large volumes of dilute aqueous solutions of heavy metalsusing this process proceed by the following steps: (1) loading of a CBRunit with a population of cells; (2) saturation of the immobilizedpopulation with the contaminant heavy metal as the dilute solution ispassed through the CBR; (3) pelleting and removal of the microorganismssaturated with heavy metal complex from the biocatalyst immobilizationchamber(s); and (4) repetition of steps 1-3 until the entire volume ofcontaminated liquid was processed.

EXAMPLE II Production of Secreted Products from Microbial Cells

Certain microbial populations have been shown to be capable of theproduction and secretion of organic molecules when these populations arepresented with a nutrient media that will support their viability. Insome cases, such nutrient media are supplemented with a stimulatorychemical that supports enhanced production of secretory products.

Microbial secretory production would be much cheaper and simpler if adense population of the productive microorganism could be maintained ina true chemostat, i.e. in invariant chemical conditions, while secretoryproducts and metabolites were continually removed from the immobilizedcellular aggregate, such a process has heretofore not been realizable.

A microbial population was immobilized in biocatalyst immobilizationchamber(s). These chambers are placed into one embodiment of the presentinvention, preferably the first or second embodiments, referred to as aCBR. The flow rate and rotor RPM were chosen to allow the immobilizationof a three dimensional array of the chosen microorganism. It isessential that the pumped system has only two phases (liquid and solid),thus the pumped system was maintained at hydraulic pressures aboveambient by means of a system pressure regulator downstream of thebiocatalyst immobilization chambers. Nutrient minerals, organics, anddissolved gas(es) were supplied to the biocatalyst immobilizationchamber by the main system pump, Pump 3.

Populations of Aureobasidium pullulans, an aerobic yeast, wereimmobilized and cultured in biocatalyst chambers using the followingparameters: RPM=380, Flow Rate=1.0 mL/min, T=22° C., pH=7.1, media: 1%glu, 1% YNB. The immobilized yeast population secreted the enzymexylanase in response to the introduction of 1% xylose into its nutrientmedia and the withdrawal of nutrient glucose. FIG. 47 shows the timecourse of xylanase production from an A. pullulans culture initiallygrown up on glucose and subsequently switched (at T=0) to xylose as themedia carbon source. Continuous production of xylanase was observed formore than 96 hours.

A comparison of the production of xylanase in a CBR unit at 25 hrs ofculture to an equivalent shake flask at 20 hrs of culture resulted inthe determination of xylanase levels of 1.2 U/mL (CBR) and 1.0 U/mL(shake flask) with the CBR-produced enzyme having ca. twice the specificactivity of the shake-flask culture. This organism is of particularinterest since the sequence of the xylanase gene and its promoter regionis known and appears to be usable for the production and secretion ofxenoproteins.

EXAMPLE III Production of Secreted Products from Animal Cells

Certain animal cell populations have been shown to be capable of theproduction and secretion of organic molecules when these populations arepresented with a nutrient media that will support their viability. Insome cases, such nutrient media are supplemented with stimulatorychemicals that supports enhanced production of secretory products. Thisexperiment shows a novel production process which exploits these animalcell characteristics to enhance the quantity and purity of secretoryproducts.

Animal cell secretory production would be much cheaper and simpler if adense population of the productive microorganism could be maintained ina true chemostat, i.e. in invariant chemical conditions, while secretoryproducts and metabolites were continually removed from the immobilizedcellular aggregate, such a process has heretofore not been realizable.The process demonstrated herein is the first demonstration of a scalablechemostatic production process for cultured animal cells.

In this new process, an animal cell population was immobilized inbiocatalyst immobilization chamber(s). These chambers were placed intoone embodiment of the present invention, preferably the first or secondembodiment. The flow rate and rotor RPM were chosen to allow theimmobilization of a three dimensional array of the chosen cell type.Since it is essential that the pumped system have only two phases(liquid and solid), the pumped system is maintained at hydraulicpressures above ambient by means of a system pressure regulatordownstream of the biocatalyst immobilization chambers. Nutrientminerals, organics, and dissolved gases are supplied to the biocatalystimmobilization chamber by the main system pump, Pump 3.

Murine hybridoma pAB122 cells were grown at 1×10⁶ cells/ml inconventional T flasks. Approximately 100 mls were removed, chilled andthe cells were pelleted by centrifugation. The cell pellet wasresuspended in 10 ml of ice cold DMEM+10% FBS and injected into thebiocatalyst chamber through an inlet while the CBR was running at theconditions RPM=250, FR=1.0 mL/min, T=37° C., pH=7.2, media: DMEM w/10%FBS. The cells were immobilized within the chamber and withinapproximately 1 hour, the initial collections of output liquid showedthe presence of antibody.

We found that the murine hybridoma pAB 122 was easily cultivable in CBRunits (RPM=250, FR=1.0 mL/min, T=37° C., pH=7.2, media: DMEM w/10% FBS).This cell line produces and secretes an antibody to the importantprotein, p53. We observed continuous production of Ab_(p53) from CBRcultures of ca. 1×10⁸ cells over 5-9 day culture periods. We found thatthe CBR-immobilized cells produced ca. 60 mg/day of Ab_(p53)continuously over a 3-4 day period. In comparison, a 7-day T-flaskculture (50 mL) of ca. 1×10⁸ cells produced ca. 7 mg/day of Ab_(p53).

In a single 3-day experiment in a CBR in which these cells were perfusedwith media lacking FBS, the production rate of Ab_(p53) decreased byonly one-half, although the time course of this latter productionappears to decrease with time. The identity of the product proteinproduced in the CBR production as Ab_(p53) has been confirmed by Westernblots, p53 ELISA assays, and by its co-migration with authentic Ab_(p53)in SDS gels.

EXAMPLE IV Bioremediation of Low Concentration Contaminants from LargeVolume Aqueous Solutions

Microbial populations have been shown to be capable of either adsorbing,absorbing, or metabolizing a wide range of organic or inorganiccompounds presented to the microbial population in dilute aqueoussolution. Further, it has been demonstrated that virtually allterrestrial, as well as many marine, microorganisms exist in nature byattachment to a solid support through the agency of either homogeneousor heterogeneous biofilms. We demonstrate herein a novel bioremediationprocess which exploits these microbial characteristics to remove watercontaminants which are presented at low concentration in very largevolume aqueous solution.

Microbial bioremediation of aqueous contaminants would be much cheaperand simpler than current remediation techniques, except that the costsof processing the dilute contaminants is prohibitive. The high flowrates which are typically required to deal with dilute contaminants will“wash out” the desired microbial population well before they can performthe desired bioremediation.

A microbial population, either homogeneous or heterogeneous, wasimmobilized on a solid support by the formation of biofilms. These solidsupports were placed into one embodiment of the present invention,preferably the third embodiment, an example of which is shown in FIG.45. The size and density of the solid support as well as the tankdimensions were chosen to allow the system pump to achieve the desiredthroughput flow rate with the generation of an immobilizedthree-dimensional array of the solid support-microbial cell complexes.Since it is essential that the pumped system has only two phases (liquidand solid), the pumped system was maintained at hydraulic pressuresabove ambient by means of a system pressure regulator downstream of thebiocatalyst immobilization chamber. Nutrient minerals, organics, anddissolved gases were supplied to the biocatalyst immobilization chamberby the main system pump, Pump 3.

The system of FIG. 45 was used to remove nitrate ion from a diluteaqueous solution, a process of great interest in environmentalbioremediation. A biocatalyst immobilization chamber having thefollowing dimensions (R₁=5.0 cm, R₂=5.1 cm, L=100 cm) was loaded with100 mL of 30-50 mesh charcoal that had been equilibrated with ashake-flask ferment of Pseudomonas putida (strain PRS2000). The mediareservoir 1 was loaded with an aqueous solution of 400 ppm sodiumnitrate, 0.05 ppm potassium phosphate, and 0.5 ppm ethanol. Thegas-liquid adsorption reservoir, 2, was equilibrated with ambientpressure nitrogen gas. The system pressure regulator, 3, was set at 30psig and Pumps 1-3 set to flow at 120 mL/min. FIG. 48 depicts the resultof an analysis of the input vs. the output levels of nitrate ion asmeasured amperiometrically. Nitrate levels in the system output fellprecipitously in the first 8 hours and remained at less than 15% of theinput value for the balance of the experiment. Collateral analysis ofthe output flow indicated that levels of nitrite and nitrous oxide wereminimal and the bulk of the nitrate had been converted to molecularnitrogen.

EXAMPLE V Serial Configurations for CBRs

Another arrangement of the present invention that is shown in FIG. 49.The present invention comprises use of several embodiments, orindividual CBRs used in serial configurations. The system configurationof FIG. 49 employs one CBR embodiment (shown in the figure as “CBR UNIT#1”) to generate ethanol by, for example, anaerobic fermentation ofglucose to ethanol by an immobilized fermentative yeast population. Theethanol so produced is then pumped into the downstream BiocatalystImmobilization Chamber where, as in Example IV above, it serves as aco-substrate for the dissimulatory reduction of nitrate ion. Notefurther that there can be a wide disparity in the throughput andcapacity of each of the serially-connected CBR units. It was shown inExample IV that dissimulation of 400 ppm nitrate required theco-presence of only 0.5 ppm ethanol. In cases such as this, CBR Unit #1is configured to immobilize a biomass that would be one thousandth ofthat immobilized in the second unit and would flow at a correspondinglysmaller flow rate.

Another arrangement of the present invention is shown in FIG. 50. Thesystem configuration of FIG. 50 employs one CBR embodiment (shown in thefigure as “CBR UNIT #1”) to generate replacement microbial cells forperiodic introduction into a parallel array of biocatalystimmobilization chambers (“Modules” in FIG. 50). The configuration ofFIG. 50 contains an array of parallel biocatalyst chambers, also calleda “Module Farm”, which is identical to the configuration of FIG. 49,except that the System Pump is, in this example, supplying contaminatedwater to four running biocatalyst immobilization chambers (Modules inFIG. 50) while two additional off-line modules are being prepared forservice.

The foregoing description of the present invention is not intended to beexhaustive or to limit the invention to the precise form disclosed. Manymodifications and variations are possible. The particular embodimentsdescribed are intended to best explain the principles of the inventionand its practical application to thereby enable others skilled in therelevant art to best utilize the present invention in variousembodiments and with various modifications as are suited to theparticular use contemplated. The foregoing description is intended tocover in the appended claims all such modifications, variations, andchanges as fall within the scope of the process of this invention.

What is claimed is:
 1. A method of producing secreted products from abiocatalyst comprising containing the biocatalyst in at least onechamber in a centrifugal force field wherein a continuous flow of aliquid acts to create a force which opposes the centrifugal force fieldand wherein a gravitational force contributes to the resultant vectorsummation of all forces acting on the biocatalyst, wherein thegravitational force, the centrifugal force and the opposing liquid forcesubstantially immobilize the biocatalyst at a position in the chamber,the liquid is at hydraulic pressures greater than ambient pressure, andwherein no gaseous phase is present in said chamber, wherein thebiocatalyst produces secreted products.
 2. The method of claim 1,wherein containing the biocatalyst comprises a plurality of chambers. 3.The method of claim 1, wherein the liquid contains a dissolved gas. 4.The method of claim 1, wherein the biocatalyst is a cell.
 5. The methodof claim 1, wherein the biocatalyst is chosen from the group comprisinga subcellular component, an enzyme complex, and an enzyme compleximmobilized on a solid support.
 6. The method of claim 1, wherein thebiocatalyst is chosen from the group comprising prokaryotic cells, algaecells, plant cells, yeast cells, fungal cells, insect cells, reptilecells and mammalian cells.
 7. The method of claim 1, wherein the liquidcontains a dissolved gas chosen from the group comprising air, O₂, CO₂,methane, NH₃, NO₂, Ar, He, N₂ and H₂ or any mixture thereof.
 8. A methodof removing contaminants from a liquid comprising containing abiocatalyst in at least one chamber in a centrifugal force field whereina continuous flow of the liquid acts to create a force which opposes thecentrifugal force field and wherein a gravitational force contributes tothe resultant vector summation of all forces acting on the biocatalyst,wherein the gravitational force, the centrifugal force and the opposingliquid force substantially immobilize the biocatalyst at a position inthe chamber, the liquid is at hydraulic pressures greater than ambientpressure, and wherein no gaseous phase is present in said chamber,wherein the biocatalyst removes contaminants from the liquid.
 9. Themethod of claim 8, wherein containing the biocatalyst comprises aplurality of chambers.
 10. The method of claim 8, wherein the liquid isan aqueous solution.
 11. The method of claim 8, wherein the liquidcontains a dissolved gas.
 12. The method of claim 8, wherein the liquidcontains a dissolved gas chosen from the group comprising air, O₂, CO₂,methane, NH₃, NO₂, Ar, He, N₂ and H₂ or any mixture thereof.
 13. Themethod of claim 8, wherein the biocatalyst is a cell.
 14. The method ofclaim 8, wherein the biocatalyst is chosen from the group comprising asubcellular component, an enzyme complex, and an enzyme compleximmobilized on a solid support.
 15. The method of claim 8, wherein thebiocatalyst is chosen from the group comprising prokaryotic cells, algaecells, plant cells, yeast cells, fungal cells, insect cells, reptilecells and mammalian cells.
 16. The method of claim 8, wherein thecontaminants are chosen from the group comprising heavy metals andnitrate ions.